WO2021174894A1 - Nanobowl-supported drug-loaded liposome, preparation method therefor, and application thereof - Google Patents

Abstract

A nanobowl-supported drug-loaded liposome, a preparation method therefor, and an application thereof. The preparation method for the nanobowl-supported drug-loaded liposome comprises: incubating and ultrasonically treating a nanobowl and a liposome and then successfully encapsulating a drug by utilizing an ammonium sulfate active drug loading method to obtain the nanobowl-supported drug-loaded liposome. The nanobowl-supported drug-loaded liposome can resist the influence of plasma proteins and blood flow shearing forces on drug leakage, enhance the delivery of the drug at a tumor site, improve carrier stability, and improve an anti-tumor curative effect.

Description

Nanobowl supporting drug-loaded liposomes and preparation method and application thereof Technical field

The invention relates to the technical field of medicine, in particular to a nanobowl supported drug-loaded liposome and a preparation method and application thereof.

Background technique

Cancer, or malignant tumor, has now become the second leading cause of death in the world and is a serious threat to human life and health. On February 3, 2017, the latest data released by the World Health Organization showed that 8.8 million people die of cancer each year in the world, accounting for nearly one-sixth of the total annual deaths in the world, and there are more than 14 million new cancer cases each year. This number will increase to more than 21 million in 2030. How to break through the world problem of cancer has become a hot issue that the life science field is eager to solve.

However, for the treatment of cancer, due to its own toxicity to normal tissue cells and extremely fast clearance rate, its efficacy is greatly limited. The birth of nano-carrier technology, due to its good biocompatibility, ideal long cycle time and accurate targeting, enables its combination with cancer treatment to break through the limitations of the drugs themselves and exert better curative effects.

The reason why nanomedicine can achieve so many tasks that a single drug cannot accomplish, one of the important mechanisms of action is enhanced permeability and retention (Enhanced Permeation and Retention, EPR effect for short). The EPR effect refers to the phenomenon that some macromolecular substances of specific sizes (such as liposomes, nanoparticles and some macromolecular drugs) are more likely to penetrate into tumor tissues and stay for a long time (compared with normal tissues). The therapeutic strategy of nanomedicine based on the EPR effect is to maximize the peak concentration (Cmax) of the drug by changing the pharmacokinetics and biodistribution of the drug, and at the same time increase the drug concentration-time curve of the drug in the plasma and tumor tissues. Area under (AUC) to improve drug efficacy and body tolerance, thereby prolonging the drug treatment level of the drug at the target site.

Among them, liposomes, as the first nanoparticle approved to be marketed for anti-tumor therapy, together with other nanoparticles designed on the basis of liposomes, account for a considerable proportion of clinical nanotherapy. However, although encapsulating chemotherapeutics in liposomes can improve the drug’s metabolic kinetics and biological tissue distribution, compared with traditional chemotherapeutics, currently marketed liposomal pharmaceutical preparations have not significantly improved the overall Survival rate. It can be seen that liposome drug delivery systems are still facing difficulties and challenges.

Among the many challenges, the stability of liposomes is very important, which determines the drug loading, leakage rate, and drug release rate of the drug during the preparation, storage, and metabolism of drug-loaded liposomes. Change. The main component of liposomes is phospholipids, and part of the phospholipids contains unsaturated fatty acid branched chains. Such phospholipids are prone to oxidative hydrolysis, resulting in a decrease in the fluidity of the phospholipid bilayer, an increase in permeability, agglomeration, and drug leakage. Increase, decrease in drug loading, etc. Studies have shown that the properties of liposomes formed by different phospholipid components will change accordingly, which will affect the stability and drug release rate of liposomes. The phase transition temperature, as another important factor, affects the storage stability of liposomes. When the temperature exceeds the phase transition temperature of the phospholipid, the phospholipid will change from a gel state to a liquid crystal state, which will loosen the lipid bilayer structure, decrease the stability, release the drug, and shorten the storage period. In addition, thermosensitive liposomes are also based on the principle of phase transition temperature, which achieves the purpose of controlling drug release by changing the temperature.

The stability of liposomes in the body is undoubtedly the key to determining the efficacy of drugs. Obviously, the EPR effect theory alone may not fully explain the series of changes that occur in the body of liposomes. After the nanoparticles are injected intravenously and before they reach the tumor tissue, they need to go through many biological processes, such as the fluid dynamics of the blood circulatory system, the interaction with various proteins and cytokines, and the tissue penetration of the nanoparticles. Changes in the shear force in the blood stream can change the degree of deformation of liposomes and affect the stability of liposomes. Studies have pointed out that high-density lipoproteins in serum can bind to phospholipids to form pores; liposomes It can activate the complement system and form a hydrophilic channel on the surface of liposomes, which leads to increased permeability of liposomes and a large amount of leakage of contents; serum albumin can even bind to liposomes to form a "crown" structure, which affects liposomes At the same time, the mononuclear phagocyte system in the circulatory system can quickly recognize liposomes and quickly clear them. For this reason, how to improve the composition, shape, size, Zeta potential, and surface properties of liposomes to overcome the above-mentioned dilemmas and to better exert the efficacy of liposome drugs is very important.

The present invention aims to establish a new type of liposome drug delivery system based on the challenges faced by liposome drug preparations at the present stage---nano bowl supporting adriamycin liposomes. Due to its unique morphology and structure, the nanobowl not only gives the liposomes sufficient rigidity and support, but also reserves enough internal cavity space for the successful loading of adriamycin. Adriamycin liposomes, as the first nanomedicine on the market, after years of clinical application, although the treatment of tumor patients has been improved to some extent, it has also exposed some of its own shortcomings. The addition of the nanobowl is expected to solve some practical problems, and has extremely high scientific value and potential clinical translational significance. The present invention investigates the different effects of nano-bowl-supported adriamycin liposomes on tumor tissues and normal tissues, thereby comprehensively evaluating the anti-tumor efficacy of the nano-bowl-supported adriamycin liposomes in vivo.

There is no report about the nanobowl supported drug-loaded liposome of the present invention and its preparation method and application.

Summary of the invention

The first objective of the present invention is to provide a nanobowl supporting drug-loaded liposome in view of the shortcomings of the prior art.

The second object of the present invention is to provide a method for preparing the drug-loaded liposome supported by the nanobowl as described above in view of the deficiencies of the prior art.

The third objective of the present invention is to provide the use of the nanobowl to support drug-loaded liposomes as described above in view of the deficiencies of the prior art.

The fourth objective of the present invention is to provide a nanobowl supported adriamycin/irinotecan/vincristine liposome in view of the deficiencies of the prior art.

The fourth objective of the present invention is to provide a preparation method of nanobowl supported adriamycin/irinotecan/vincristine liposomes in view of the shortcomings of the prior art.

In order to achieve the above-mentioned first objective, the technical solution adopted by the present invention is:

The nanobowl supports drug-loaded liposomes, and the preparation method thereof includes the following steps:

(1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

(2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

(3) Preparation of nanobowl-supported drug-loaded liposomes: using the method of active drug loading of ammonium sulfate to encapsulate the drug, the nanobowl-supported drug-loaded liposomes are obtained.

Preferably, the drug in step (3) is selected from any one of doxorubicin, irinotecan, and vincristine.

In order to achieve the above-mentioned second objective, the technical solution adopted by the present invention is:

The preparation method of nanobowl supported drug-loaded liposomes as described above includes the following steps:

(1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

(2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

(3) Preparation of nanobowl-supported drug-loaded liposomes: using the method of active drug loading of ammonium sulfate to encapsulate the drug, the nanobowl-supported drug-loaded liposomes are obtained.

In order to achieve the above-mentioned third objective, the technical solution adopted by the present invention is:

The nanobowl supports the application of drug-loaded liposomes in the preparation of anti-tumor drugs.

In order to achieve the fourth objective mentioned above, the technical solution adopted by the present invention is:

The nanobowl supports adriamycin/irinotecan/vincristine liposomes, and its preparation method includes the following steps:

(1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

(2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

(3) Nanobowl supported doxorubicin/irinotecan/vincristine liposome preparation: use ammonium sulfate active drug loading method to encapsulate doxorubicin/irinotecan/vincristine to obtain nanobowl support Adriamycin/irinotecan/vincristine liposomes.

Preferably, it is characterized in that the particle size of the polystyrene nanoparticles in step (1) is 45-55nm; the peanut-shaped nanoparticles are made of styrene monomer and MPS modified polystyrene nanoparticles according to Vstyrene:VMPSNPs=3:1 prepared; the silica-modified peanut-shaped nanoparticles are prepared with 0.3g TEOS.

Preferably, it is characterized in that the average particle size of the nanobowl supported adriamycin/irinotecan/vincristine liposome is 140-150 nm, and the Zeta potential is -18 to -16 mV.

In order to achieve the above-mentioned fifth objective, the technical solution adopted by the present invention is:

The preparation method of nanobowl supported adriamycin/irinotecan/vincristine liposomes as described above includes the following steps: (1) Preparation of nanobowl: preparation of polystyrene nanoparticles → preparation of MPS coating modification Polystyrene nanoparticles→preparation of peanut-shaped nanoparticles→preparation of silica modified peanut-shaped nanoparticles→obtain nano bowls;

(2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

(3) Nanobowl supported doxorubicin/irinotecan/vincristine liposome preparation: use ammonium sulfate active drug loading method to encapsulate doxorubicin/irinotecan/vincristine to obtain nanobowl support Adriamycin/irinotecan/vincristine liposomes.

Preferably, the preparation method of the nanobowl in step (1) is:

1) Preparation of polystyrene nanoparticles: using styrene as a monomer, SDS as an emulsifier, and KPS as an initiator, the polystyrene nanoparticles are synthesized by emulsion polymerization;

2) Preparation of MPS-coated modified polystyrene nanoparticles: on the basis of synthesized polystyrene nanoparticles, styrene, MPS and AIBN are added, and MPS-coated modified polystyrene nanoparticles are synthesized through polymerization;

3) Preparation of peanut-like nanoparticles: Mix the prepared MPS-coated modified polystyrene nanoparticles with styrene and VBS, and put them in ultrapure water to stir. The swelling effect of polystyrene is used to make MPS The coated modified polystyrene nanoparticles are deformed and broken, and then AIBN is added to initiate the polymerization reaction again, and finally form peanut-shaped nanoparticles;

4) Preparation of silica-modified peanut-shaped nanoparticles: the peanut-shaped nanoparticles obtained in 3) were ultracentrifugated and then dispersed again in absolute ethanol, and then 25% concentrated ammonia was added, and the configuration contained 50% TEOS After slowly dripping the ethanol solution of, the peanut-shaped nanoparticles modified by silica are obtained;

5) Preparation of nanobowl: transfer the silica-modified peanut-like nanoparticles obtained in 4) to a rotary evaporator, after evaporating excess ethanol, add tetrahydrofuran to dissolve, collect and precipitate by ultracentrifugation to obtain the final product-nanobowl .

Preferably, the particle size of the polystyrene nanoparticles in step (1) is 50 nm; the peanut-shaped nanoparticles are modified polystyrene nanoparticles with styrene monomer and MPS according to V _styrene :V _MPSNPs = 3:1 prepared; the silica-modified peanut-shaped nanoparticles are prepared with 0.3 g of TEOS.

The nanobowl prepared by the present invention supports the adriamycin liposome through the supporting effect of the nanobowl, which provides a "hard" inner bladder for the liposome, so that it can withstand the impact and destruction of various factors in the circulatory system , Before reaching the tumor site, minimize the leakage of the contents of the drug, so that more doxorubicin can reach the tumor site with the circulatory system, and improve the stability of the active drug-loaded liposomal doxorubicin (DOX). So as to better exert the efficacy of drugs.

The advantages of the present invention are:

1. The drug-loaded liposomes supported by nanobowl prepared by the present invention can resist the influence of plasma protein and blood flow shear force on drug leakage. This method improves the delivery of drugs to tumor sites and improves the efficacy of anti-tumor. Compared with other methods that increase stability by changing the liposome bilayer and composition, this method designs a physical support for the cavities of all-water nanoliposomes. Nanobowl stabilized liposomes improve carrier stability and drug release.

2. The present invention overcomes the shortcomings of the prior art (due to the leakage of drugs in the blood circulation, liposome drug delivery for cancer treatment may be restricted), by embedding a hard nanobowl in the liposome water cavity To improve the stability of active drug-loaded liposomal drugs, optimize the types of raw materials and their ratios and process parameters, and obtain the nanobowl-supported drug-loaded liposomes with the best curative effect, and nanobowl support The drug-loaded liposomes can resist the influence of plasma protein and blood flow shear force on drug leakage, improve the delivery of drugs to tumor sites, improve the anti-tumor efficacy and have no toxic side effects, and change the liposome bilayer, Compared with other methods to enhance stability, such as composition, this method designs physical support for the cavity of full-water nanoliposomes, improves the survival rate of breast cancer patients, and reduces the economic burden of such patients. Application prospects.

Description of the drawings

Figure 1 is a schematic diagram of the nanobowl synthesis circuit.

Figure 2 is: (AE) are the DLS particle size distribution diagrams of polystyrene nanoparticles, MPS-coated modified polystyrene nanoparticles, peanut-shaped nanoparticles, silica-modified peanut-shaped nanoparticles, and nanobowls. ; (FJ) are corresponding to polystyrene nanoparticles, MPS-modified polystyrene nanoparticles, peanut-shaped nanoparticles, silica-modified peanut-shaped nanoparticles and nanobowls respectively.

Figure 3 is a transmission electron micrograph of (A) polystyrene nanoparticles, (B) peanut-like nanoparticles and (C) silica modified peanut-like nanoparticles.

Figure 4 is: (A, B) are the TEM images of the nanobowl, (C) is the opening angle of the nanobowl estimated based on the radius and the aspect ratio, and (D-G) are the nanobowls with different angles under the TEM.

Fig. 5 is: (A) is a transmission electron microscope image of small-sized polystyrene nanoparticles, (B, C) is a diagram of particle size distribution and Zeta potential of small-sized polystyrene nanoparticles, respectively.

Fig. 6 is: (A) is a transmission electron microscope image of peanut-shaped nanoparticles synthesized with a feeding ratio of 3:1, and (B) is a transmission electron microscope image of peanut-shaped nanoparticles synthesized with a feeding ratio of 9:1.

Figure 7 is: (A) is a transmission electron microscope image of a nanobowl synthesized with 0.3g TEOS, and (B) is a transmission electron microscope image of a nanobowl synthesized with 0.5g TEOS.

Figure 8 is a schematic diagram of a nanobowl supported adriamycin liposome synthesis circuit.

Fig. 9 is: (A) is a transmission electron microscope image of the aminated nanobowl, (B, C) are the particle size distribution and zeta potential of the aminated nanobowl, respectively.

Figure 10 is: (A) is a schematic diagram of nanobowl supporting liposomes, (B) is a transmission electron microscope image of nanobowl supporting liposomes, (C, D, E, F) are nanobowl supporting liposomes, respectively And ordinary liposome particle size distribution and Zeta potential diagram.

Figure 11 is: (A) is the ultraviolet absorption and fluorescence spectrum of doxorubicin hydrochloride, (B) is the ultraviolet absorption and fluorescence spectrum of the DiR probe.

Fig. 12 is: (A) is the ultraviolet absorption and fluorescence spectra of nanobowl-supported adriamycin liposomes, and (B) is the ultraviolet absorption and fluorescence spectra of ordinary adriamycin liposomes.

Figure 13 is a schematic diagram of ammonium sulfate active drug loading.

Figure 14 is: (A) the drug leakage rate of common doxorubicin liposomes and nanobowl supported doxorubicin liposomes in whole serum; (B) nanobowl supported liposomes in serum within 24 hours of particle size And Zeta potential change, n=3.

Figure 15 is: (A, D) are the nanobowl supported adriamycin liposomes and ordinary adriamycin liposomes and then dispersed after freeze-drying, (B, C, E, F) are nano bowls respectively The particle size distribution comparison chart of supporting adriamycin liposome and ordinary adriamycin liposome before and after freeze-drying.

Fig. 16 is a graph showing the particle size distribution and zeta potential change of nanobowl supported adriamycin liposomes during storage at 4°C, n=3.

Figure 17 is: (A) the tumor cell uptake of nanoparticles taken under a laser confocal microscope, Bar=25m; (B) the quantitative analysis of the fluorescence intensity of the cells uptake of the nanoparticles, the result is represented by "mean±SD", n= 5. *P<0.05, **P<0.01, ***P<0.001.

Figure 18 is: (A) the effect of free adriamycin hydrochloride, ordinary adriamycin liposomes, nanobowl supported adriamycin liposomes on the viability of 4T1 breast cancer cells, (B) unloaded blank nanobowl The effect of supporting liposomes on the viability of 4T1 cells is expressed by mean±SD, n=4. **P<0.01, ***P<0.001.

Figure 19 is a diagram showing the effect of nanobowl supported adriamycin liposomes for treating BALB/c female mice bearing 4T1 breast cancer; (A) schematic diagram of administration; (B, C) respectively the charge of each administration group Tumor volume of tumor mice and mouse body weight, n=8; In Figure B, a is the statistical difference between the tumor volume of mice in the free adriamycin and normal saline and blank carrier groups, and b is the common adriamycin lipid The statistical difference between the tumor volume of mice in the body and the normal saline and the blank carrier group, c is the statistical difference between the tumor volume of the mice in the nanobowl supported adriamycin liposome and the other four groups of experimental groups; (D) The survival curve of tumor-bearing mice in each administration group, *P<0.05, **P<0.01, ***P<0.001.

Figure 20 is (A) 4T1 tumor-bearing mice tumor histopathological section and immunohistochemical staining analysis, Bar=100m, (B, C) are the statistics of TUNEL and PCNA positive rates of tumor tissues after treatment in each experimental group; experiment; The results are expressed by mean±SD, n=5, *P<0.05, **P<0.01, ***P<0.001.

Figure 21 is an immunohistochemical staining analysis of important organs in 4T1 tumor-bearing mice, Bar=100m.

Figure 22 is: (A) ordinary irinotecan liposomes and nanobowl supported irinotecan liposomes and (B) ordinary vincristine liposomes and nanobowl supported vincristine liposomes in FBS Drug leakage stability.

Detailed ways

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The abbreviations described in the present invention are shown in Table 1.

Table 1. List of acronyms

Example 1 Construction and Characterization of Nanobowl

1. Construction of Nano Bowl

1 Instruments and materials

1.1 Instrument and equipment

Table 2

XS205s electronic balance	METTLER TOLEDO
HS-70 type thermostatic magnetic stirrer	German IKA company
HD2010W Constant Speed Electric Stirrer	Shanghai Sile Instrument Co., Ltd.
BT100-2J peristaltic pump	Baoding Lange Constant Flow Pump Co., Ltd.
R-200 Rotary Evaporator	Swiss Buchi Company
Sorvall ST 16 refrigerated centrifuge	Thermo Fisher Scientific
Optima TM XPN ultracentrifuge	American Beckman Coulter Co., Ltd.
CM-120 Transmission Electron Microscope	Philips Netherlands
Malvern Zetasizer Nano ZS Laser Particle Sizer	Malvern Company
MS2 type vortex mixer	German IKA company

Laboratory ultrapure water system

Millipore Corporation

1.2 Materials and reagents

table 3

Styrene	American Sigma Company
SDS	Shenggong Biological Engineering (Shanghai) Co., Ltd.
KPS	Shanghai Aladdin Chemical Reagent Co., Ltd.
MPS	American Sigma Company
AIBN	Shanghai Aladdin Chemical Reagent Co., Ltd.
VBS (90%)	American Sigma Company
TEOS	American Sigma Company
Absolute ethanol	Sinopharm Chemical Reagent Co., Ltd.
25% concentrated ammonia	Shanghai Aladdin Chemical Reagent Co., Ltd.
Tetrahydrofuran	Sinopharm Chemical Reagent Co., Ltd.

2 Experimental method

2.1 Synthesis of polystyrene nanoparticles

The emulsion polymerization method was used to synthesize polystyrene nanoparticles with a size of 50 nm. Specific steps are as follows:

1) Precisely weigh 1.00 g of sodium dodecyl sulfonate and place it in a clean 250 mL three-necked flask;

2) Use a 100mL graduated cylinder to accurately measure 85mL of ultrapure water and pour it into a 250mL three-necked flask containing sodium dodecyl sulfonate;

3) Connect a spherical condenser and two rubber stoppers to the three mouths of the three-necked flask;

4) Insert a needle into the rubber stopper of one of the mouths of the three-necked flask, so that the needle is completely submerged below the liquid surface, and at the same time connect a catheter to the other end of the needle, and the other end of the catheter is connected to argon;

5) Open the valve of the argon cylinder, pour argon into the three-necked flask, adjust the valve, control the flow rate of argon, and observe even bubbling in the liquid surface of the three-necked flask;

6) At the same time, keep stirring at 250rpm for 1h by HS-70 type thermostatic magnetic stirrer;

7) Remove the needle from the three-necked flask, and switch the argon-filled tube to the spherical condenser to continue to ventilate;

8) Precisely pipette 20.00g styrene monomer, insert a rubber stopper with a 50mL syringe, inject it into the three-necked flask, and continue to keep magnetic stirring for 30 minutes;

9) Turn on the heating switch of the thermostatic magnetic stirrer and adjust the temperature to heat the reaction system to 70°C;

10) Precisely weigh 0.10 g of potassium persulfate, add it to 10 mL of ultrapure water, and vortex for 1 min with an MS2 vortex mixer to make it fully dissolved;

11) After the temperature of the reaction flask is equilibrated, use a 10mL syringe to slowly add the pre-prepared potassium persulfate solution drop by drop;

12) Under the protection of argon, the reaction system was continuously stirred for 18 hours and then the reaction was stopped to obtain a polystyrene nanoparticle emulsion;

13) During the reaction process, the liquid in the three-necked flask should gradually change from a thinner and blue-blue emulsion to a thicker milky white liquid;

14) After stopping the reaction, collect the white emulsion of the product and transfer it to a dialysis bag with a molecular weight cut-off of 10KD. Use ultrapure water as the dialysis medium for dialysis. Change the dialysis medium at least 3 times to remove residual styrene and dodecane. Sodium sulfonate

15) After the dialysis, transfer 1.00 g of liquid into an oven with constant weight, and determine the mass concentration of the polystyrene nanoparticle emulsion.

2.2 Copolymerization of styrene and 3-(trimethoxysilyl) propyl methacrylate

Through the copolymerization of styrene and 3-(trimethoxysilyl) propyl methacrylate, the surface of the hydrophobic polystyrene nanoparticles is covered with a relatively hydrophilic polymer layer. The experimental steps are as follows:

1) Dilute the milky white emulsion with ultrapure water to a mass concentration of 10%;

2) Transfer the diluted emulsion to a new 100mL three-necked flask, and connect the spherical condenser, HD2010W constant-speed electric stirrer and a rubber stopper to the three mouths of the flask;

3) Insert a needle into the rubber stopper of the three-necked flask, and pour argon into the three-necked flask as described in 4-5) in 2.1;

4) At the same time, keep mechanical stirring at 400rpm for 1h through HD2010W constant speed electric stirrer;

5) Remove the needle from the three-necked flask, and switch the argon-filled tube to the spherical condenser to continue to ventilate;

6) At a volume ratio of 4:1, styrene and 3-(trimethoxysilyl) propyl methacrylate are uniformly mixed, and the MS2 type vortex mixer is fully vortexed to obtain a mixed monomer solution and mix The total mass of the monomer solution is equivalent to the mass of the polystyrene nanoparticles in the above emulsion;

7) Add the resulting mixed monomer solution to the reaction flask through a 10 mL syringe, and continue to stir for 1 hour;

8) Subsequently, add azobisisobutyronitrile equivalent to 3% of the mass of the mixed monomer solution as a reaction initiator, and continue to stir and mix for 1 hour;

9) Turn on the HS-70 thermostatic magnetic stirrer, heat to 70°C, stop the reaction after 18 hours of reaction, and obtain MPS modified polystyrene nanoparticles;

10) After the reaction, as described in section 13-14) in 2.1, perform dialysis and constant weight to determine the mass concentration of the MPS modified polystyrene nanoparticle emulsion;

11) According to the measured mass concentration of the emulsion, dilute it with ultrapure water to a mass concentration of 5% to prevent aggregation.

2.3 Synthesis of peanut-like nanoparticles

The modified nanoparticle emulsion is further diluted to a mass concentration of 3.5%, and the peanut-shaped nanoparticle is obtained by further swelling with the styrene monomer. Specific steps are as follows:

1) Weigh the sodium p-styrene sulfonate equivalent to 0.8% of the nanoparticle mass, add it to a 100mL three-necked flask containing nanoparticle emulsion, and stir for 15min to make it uniformly dissolved;

2) Then connect the spherical condenser, HD2010W constant speed electric stirrer and a rubber stopper to the mouth of the flask;

3) Insert a needle into the rubber stopper of the three-necked flask, and pour argon into the three-necked flask as described in 4-5) in 2.1;

4) At the same time, adjust the speed of HD2010W constant speed electric stirrer to 250rpm, and keep the mechanical stirring for 1h;

5) Take styrene monomer equivalent to three times the volume of nanoparticles and 3% mass concentration of azobisisobutyronitrile and mix uniformly, and the MS2 type vortex mixer is fully vortexed to dissolve, and it is used as a swelling solution;

6) Add the above-mentioned swelling liquid to the reaction system through a 10mL syringe, and continue to stir for 1 hour to achieve full swelling;

7) Then, turn on the HS-70 constant-temperature magnetic stirrer, heat to 70°C, stop the reaction after 18 hours of reaction, and obtain a peanut-shaped nanoparticle emulsion;

8) After the reaction, the precipitate was collected by an ultracentrifuge (30000 g, 30 min), and dispersed in an equal volume of absolute ethanol by ultrasonic in a water bath. This process was repeated three times to remove residual unreacted monomer and initiator.

2.4 Synthesis of peanut-like nanoparticles modified by silica

Using the different hydrophilicity of the two hemispheres of the peanut-like nanoparticles synthesized in the previous step, the silane hydrolysis and condensation reaction only occurs on the hemisphere containing 3-(trimethoxysilyl) propyl methacrylate. The main steps are as follows:

1) Measure 4 mL of the nanoparticle ethanol dispersion obtained from the above reaction and place it in a 100 mL round bottom flask;

2) Add 6 mL of absolute ethanol to the round bottom flask to further dilute to 10 mL, and add 0.5 mL of 25% concentrated ammonia;

3) Regulate HS-70 type thermostatic magnetic stirrer to keep stirring at 500rpm;

4) Accurately weigh 0.30g of tetraethyl orthosilicate and mix it evenly with the same quality of absolute ethanol. The MS2 type vortex mixer will be fully vortexed before use;

5) Use a peristaltic pump to slowly inject the mixture of ethyl orthosilicate and absolute ethanol into the reaction solution at a flow rate of 1 mL/h. After the injection of the mixture is completed, continue to keep stirring for 1 hour;

Synthesis of 2.5 Nano Bowl

Finally, using the characteristic of polystyrene to dissolve in tetrahydrofuran, the polymer template is removed to obtain nano particles with a bowl-shaped structure. The specific experimental steps are as follows:

1) Open the water bath of the R-200 rotary evaporator and set the water temperature to 45°C;

2) Connect the round bottom flask containing the nanoparticle ethanol dispersion to the rotary evaporator after the above-mentioned reaction, adjust the rotation speed to medium speed, and keep the rotary evaporation for 1 hour to ensure that the ethanol is completely volatilized;

3) After removing the ethanol, remove the round bottom flask and add an appropriate amount of tetrahydrofuran;

4) Put the round-bottomed flask into the water bath and ultrasonic for 5min, so that the dry powder after rotary steaming can be evenly dispersed;

5) Put the round bottom flask on HS-70 type thermostatic magnetic stirrer, and keep high-speed stirring (1200rpm) for 24h;

6) Collect the reaction solution, collect the precipitate through an ultracentrifuge (30000g, 30min), and re-disperse it in three-distilled water by ultrasonic in a water bath. This process is repeated three times.

2. Calculation of the mass concentration and synthesis yield of nanoparticle emulsions obtained in each step of the reaction

Perform constant weight operation on the nanoparticle emulsion obtained from each step of the reaction:

1) Select 3 clean 1.5mL empty EP tubes, and record the initial tube weight with a constant weight in the oven;

2) Precisely weigh three 1.00g of each nanoparticle emulsion into the EP tube after constant weight, and put it into the oven;

3) Take out the EP tube regularly, weigh, record, and continue to put it back into the oven. After the final two masses do not change, the end point of constant weight will be reached;

4) Calculate the mass of the nano-particles and calculate the mass concentration through the differential weighing method. The calculation formula is as follows:

5) Calculate the yield of each synthesis step according to the calculated mass concentration, the calculation formula is as follows:

3. Nanoparticle characterization method

1. Nanoparticle DLS and Zeta characterization: After nanoparticles are dispersed in ultrapure and diluted to an appropriate concentration, the light scattering particle size and Zeta potential are measured by Malvern Zetasizer Nano ZS laser particle size analyzer.

2. The nanoparticle solution prepared in the key steps of the foregoing method is diluted and dropped on a reinforced copper mesh, and the morphology of the nanoparticle is observed through a CM-120 transmission electron microscope.

3. Estimation of the opening angle of the nanobowl

1) Measure the radius ratio (r ₁ :r ₂ ) of the two hemispheres of the peanut-shaped nanoparticles and the overall aspect ratio (L:W) of the _{peanut-shaped nanoparticles by transmission electron microscope, where W=2*r 2} .

2) According to the radius ratio and the aspect ratio, calculate the opening angle of the nanobowl after the polystyrene is dissolved. The calculation formula is as follows:

Fourth, the result

1. Mass concentration of nanoparticle emulsion

The results of the concentration of each nanoparticle emulsion calculated according to the mass concentration calculation formula are shown in Table 4. The synthesis yields are all maintained above 85%, and the synthesis efficiency is relatively high.

Table 4. Mass concentration and synthesis yield of each nanoparticle emulsion

2. Nanoparticle size distribution and Zeta potential

The particle size distribution and Zeta potential of each nanoparticle are shown in Figure 2: The polystyrene nanoparticles were successfully synthesized by the emulsion polymerization method. The average size of the polystyrene nanoparticles was 50.8nm, and the PDI was 0.068, indicating that the particle size uniformity of the nanoparticles is good, and the Zeta potential At -40.7mV. Through the copolymerization of styrene and 3-(trimethoxysilyl) propyl methacrylate, the final MPS modified polystyrene nanoparticles have an average size of 68.5nm. At the same time, due to 3-(trimethoxysilyl) The effect of silyl) propyl methacrylate makes it more negative, and the Zeta potential is -49.2mV. The modified nanoparticles and the styrene monomer were further stirred, and the swelling effect and polymerization reaction were used to obtain peanut-like nanoparticles. The average size of the nanoparticles was about 137.8nm. The increase in the content of styrene resulted in a certain degree of Zeta potential. It rose back to -31.6mV. On the basis of the above, a peristaltic pump was used to add slowly ethyl orthosilicate. After the hydrolysis and condensation reaction occurred, the size of the nanoparticles was further increased to 154.7 nm, and the Zeta potential was -39.5 mV. Finally, due to the addition of tetrahydrofuran, the polystyrene template was dissolved, and the size of the final product was 126.7nm, which was significantly reduced from the size of the previous silica-modified peanut-like nanoparticles. At the same time, the Zeta potential remains around -30.2mV.

The specific values of the particle size and Zeta potential obtained by the DLS detection of the nanoparticles in the above steps are summarized in Table 5:

Table 5. Data of particle size and surface potential detected by DLS of each nanoparticle (n=3)

3. Transmission electron micrograph of nanoparticles

The key reaction step of the nano-particles was diluted to an appropriate concentration, and the nano-particle dispersion was dropped on the carbon-supported copper mesh, and after natural air-drying, the sample was observed with a transmission electron microscope. The results are shown in Figures 3 and 4.

It can be seen from Figure 3.A that the synthesized polystyrene nanoparticles have good monodispersity, uniform size, and the size is around 50nm, which is consistent with the DLS detection results. Since the synthesis process of MPSNPs does not significantly change the morphology of nanoparticles, and the size of nanoparticles does not increase significantly, transmission electron microscopy cannot accurately prove the success of their synthesis. Therefore, this embodiment no longer adds corresponding TEM images, but uses DLS to detect particle size and Zeta potential and follow-up TEM image observation of nanoparticles to verify the synthesis result of MPSNPs.

As the reaction progresses, on the basis of MPSNPs, the swelling effect of styrene leads to a significant change in the morphology of the nanoparticles. As shown in Figure 3.B, the nanoparticles changed from a regular round sphere to two Hemispheres are connected to form a peanut-like nano-particle structure. At the same time, it can be found that because the nano particles are transformed from spheres to rod-like irregularities, the detection of their particle size should be measured by their longitudinal length and transverse width at the same time. In combination with the measurement results of the particle size in DLS, it is not difficult to find that the value is closer to the longitudinal length of the nanoparticle in the transmission electron microscope. And through the transmission electron microscope image, it can be inferred that the lateral width of the nanoparticles has little increase compared with the original polystyrene nanoparticles. The successful synthesis of peanut-shaped nanoparticles and the similarity of their width to polystyrene nanoparticles have verified the successful preparation of MPSNPs from the side.

After TEOS was introduced, the peanut-shaped nanoparticles containing MPS groups were covered with silica on the hemispheres. At the same time, due to the generation of tiny silica nanoparticles in this reaction environment, although the method of reducing the TEOS addition rate through a peristaltic pump can effectively reduce the generation of silica nanoparticles, it still cannot be completely avoided. When the tiny silica nanoparticles react with the peanut-shaped nanoparticles again, the surface of the silica-modified peanut-shaped nanoparticles is not smooth, as shown in Figure 3.C, compared to Figure 3.B. , Is rougher, and can observe tiny bumps on the surface.

Finally, under the action of tetrahydrofuran, after the polystyrene is dissolved, it can be clearly seen under the transmission electron microscope that one hemisphere of the original peanut-shaped nanoparticles has disappeared, and the nanoparticles have transformed into a sphere. It is worth noting that the center of the remaining half of the sphere has a significantly lower electron density than the periphery, showing a lighter grayish white than the periphery, indicating that the center of the sphere has also been hollowed out. Silicon oxide shell. Moreover, from the TEM images, the nanobowls with different opening orientations can be clearly observed (Figure 4). The above evidence all proves the successful synthesis of the nanobowl.

4. Radius ratio, aspect ratio and nano bowl opening angle of peanut-shaped nanoparticles

1) Radius ratio and aspect ratio of peanut-shaped nanoparticles

By measuring the length and width of the peanut-shaped nanoparticles in the transmission electron microscope, and the radius of the two hemispheres, the calculation results are r ₁ :r ₂ ≈9:10, L:W≈3:2.

2) Nano bowl opening angle

According to the measured length, width, and radius of the two hemispheres of the peanut-shaped nanoparticles, the opening angle (θ) of the nanobowl is calculated to be in the range of 90°-110° using the opening angle calculation formula.

Five, discussion

1. Selection and control of the size of polystyrene nanoparticles

As the seed template in the preparation, the subsequent reactions must be carried out on the basis of the completion of polystyrene nanoparticles. Therefore, the preparation of polystyrene nanoparticles will directly affect the success of subsequent reactions, and the synthesis of high-quality polystyrene Ethylene nanoparticles are particularly important. Since the subsequent reaction needs to use a single polystyrene nanoparticle as a template, there are higher requirements for the uniformity and monodispersity of the polystyrene nanoparticle. Any adhesion or agglomeration may affect the subsequent steps of the reaction, leading to changes in the properties of the final nanoparticles, changes in morphology, and even synthesis failure. At the same time, the choice of the size of the polystyrene nanoparticles will also affect the overall size of the final nanobowl and the volume of the internal water cavity. According to the EPR effect theory, polystyrene nanoparticles in the range of 30-50nm are finally selected as the seed template, and after a multi-step reaction, the final nanobowl has an overall size of about 100nm and has an internal water diameter of 30-50nm. Cavity. According to the characteristics of emulsion polymerization, by changing the feeding amount of the emulsifier SDS, the volume of the emulsion droplets in the reaction system can be changed, thereby affecting the volume of styrene coated in the emulsion droplets, and ultimately determining the size of polystyrene nanoparticles . This article has tried to select two different SDS dosages (1g, 2g) to synthesize polystyrene nanoparticles of different sizes. It was found that when the amount of SDS was increased, the size of the polystyrene nanoparticles was significantly reduced to around 30nm. The DLS test result showed 38.74±12.7nm, the Zeta potential was -40.9±3.1mV, but the PDI result showed 0.372± 0.008. At the same time, the polystyrene nanoparticles observed by the transmission electron microscope showed obvious agglomeration and adhesion (Figure 5). From the results of PDI and transmission electron microscopy, it can be proved that although increasing the amount of SDS can reduce the size of polystyrene nanoparticles, due to the decrease in size, the surface energy of the nanoparticles increases significantly, resulting in a significant decrease in the stability of the nanoparticles , It is easy to reunite. Therefore, after consideration, polystyrene nanoparticles with a size of 50 nm were finally selected as the seed template for the subsequent reaction.

2. The swelling effect of styrene and the synthesis principle of peanut-like nanoparticles

In the swelling process, the mixing time of styrene monomer and MPS modified polystyrene nanoparticles in advance is very important. Only by stirring for sufficient time to allow the styrene monomer to penetrate into the MPS modified polystyrene nanoparticles, can the polystyrene nanoparticles located in the center of the MPS modified polystyrene nanoparticles expand in volume. The expanded core polystyrene nanoparticles gradually ruptured the coating shell of MPS and styrene copolymer, forming a gap. Subsequently, the temperature of the reaction system is increased. Under the initiation of AIBN, the remaining styrene monomer in the reaction system will rapidly undergo explosive polymerization at the gap, forming another hemisphere of peanut-shaped nanoparticles at the gap. It is worth noting that during the polymerization reaction, the styrene monomer cannot fully contact and react with MPS due to the hydrophobicity of styrene and the hydrophilicity of MPS. With the same hydrophobicity, based on the principle of similar compatibility, styrene monomers are more likely to undergo rapid polymerization at the gap.

3. Control of the swelling ratio of styrene and the hemisphere size of peanut-like nanoparticles

By adjusting the added amount of styrene monomer, the size of one of the hemispheres produced by swelling of the peanut-shaped nanoparticles can be adjusted. In this paper, two different feed ratios of styrene monomer and MPS modified polystyrene nanoparticles were selected, 3:1 and 9:1, respectively, to verify the above argument. The results are shown in the transmission electron microscope of Figure 6, when the volume ratio of styrene monomer to MPS modified polystyrene nanoparticles (V _styrene :V _MPSNPs ) is 3:1, two of the synthesized peanut-shaped nanoparticles The hemisphere size is comparable (Figure 6.A). When Vstyrene:VMPSNPs increased to 9:1, the size of the two hemispheres of the synthesized peanut-like nanoparticles was very different (Figure 6.B), and the smaller hemisphere was the original MPS-modified polystyrene nanoparticles. However, due to the increase of styrene monomer, the size of the other hemisphere has increased significantly, and its diameter has reached 200nm, which is 4 times the size of the smaller hemisphere. In view of the need to deduct polystyrene in subsequent reactions, an excessive proportion of polystyrene may lead to incomplete dissolution process, thereby affecting the synthesis of the final nanobowl. Therefore, _{a feeding ratio of V styrene} :V _MPSNPs =3:1 was selected as the final synthesis of peanut-like nanoparticles.

4. Selective hydrolysis and condensation of TEOS and control of flow rate

After successfully synthesizing peanut-like nanoparticles, TEOS was added for hydrolysis and condensation reaction. Because the synthesized peanut-like nanoparticles are not only special in shape, but also the surface groups of the two hemispheres are completely different. Due to the addition of MPS, one hemisphere of peanut-like nanoparticles contains silanol groups, and the other hemisphere formed later is polymerized by simple styrene monomer, and its surface has only ethylenic bonds of styrene. There is no silanol group. Therefore, under the alkaline condition of ammonia water, TEOS can pass

It reacts with the silanol group on MPS to undergo hydrolysis and condensation reaction. Since there is no silanol group on the other hemisphere, the hydrolysis and condensation reaction cannot occur, so that the silica and the hemisphere containing MPS can be selectively modified. It should be noted that TEOS itself can also hydrolyze and condense under the catalysis of ammonia to form silica nanoparticles. In order to reduce the probability of TEOS self-hydrolysis and condensation, the proper TEOS addition speed is a very critical influencing factor. By reducing the TEOS addition speed as much as possible, the additional TEOS quickly reacts with the peanut-like nanoparticles, thereby reducing the silica nanoparticles formed due to their own condensation. However, although this operation can reduce the probability of the generation of silica nanoparticles, it is still inevitable to form tiny silica nanoparticles, and continue to condense with peanut-like nanoparticles in the form of tiny silica nanoparticles. Therefore, as mentioned above, the surface of the silica-modified peanut nanoparticles is not smooth.

5. The amount of TEOS and the shape of the nanobowl

Finally, this example also investigated the relationship between the TEOS feeding amount and the shape of the nanobowl. The TEOS dosages of 0.3g and 0.5g were selected to prepare nanoparticles, and the final nanobowls were obtained, and they were dropped on the copper mesh respectively, and the morphological differences of the nanobowls were observed by transmission electron microscope. As shown in Figure 7.A, when the input amount of TEOS is 0.3g, the silica layer on the surface of the nanoparticles can be clearly observed, and the structure of the bowl mouth and the bowl wall can be clearly distinguished. However, when the amount of TEOS is increased to 0.5g, due to the increase of TEOS, the probability of its own hydrolysis and condensation is also significantly increased. While the nanobowl is formed, the production of silica nanoparticles is also significantly increased, as shown in Figure 7. As shown in B, compared to Fig. 7.A, the proportion of solid silica nanoparticles is greatly increased. Therefore, in order to obtain more nano bowls, 0.3 g of TEOS is selected as the final preparation of silica modified peanut-shaped nanoparticles.

Six, summary

The construction method of the nanobowl: use styrene monomer as the raw material to synthesize polystyrene nanoparticles by emulsion polymerization; then add the MPS/styrene mixed solution mixed in a certain proportion, and prepare the MPS modified poly Styrene nanoparticles; on this basis, styrene monomer is added again, and the swelling properties of styrene and polymerization reaction are used to obtain peanut-shaped nanoparticles; the prepared peanut-shaped nanoparticles are transferred to absolute ethanol for uniform dispersion Then, TEOS is added, and under the alkaline environment of concentrated ammonia water, it selectively interacts with the hemisphere containing the MPS group.

Reaction to form silica-modified peanut-shaped nanoparticles; finally, using the characteristic of polystyrene to dissolve in tetrahydrofuran, dissolve the polystyrene component in the nanoparticles, leaving only the shell containing silica, thus obtaining Bowl-shaped structure of nanoparticles. The feeding ratio of each reaction step was determined by optimizing the conditions, and the final prepared nanobowl had an average particle size of 126.7nm, a Zeta potential of -30.2mV, a PDI of 0.142, a uniform nanoparticle size, and good monodispersity. Transmission electron microscope observation verified the morphological structure of the nanobowl; and through the radius ratio and aspect ratio of the nanoparticles, it was estimated that the opening angle of the nanobowl was in the range of 90°-110°.

Example 2 Preparation of Nanobowl Supported Liposomes and Establishment of Drug Loading Methods

In this example, the liposome and the nanobowl prepared in Example 1 were combined by electrostatic adsorption by means of probe ultrasound. Subsequently, the ammonium sulfate gradient method was used for active drug loading to complete the loading of the weakly basic drug adriamycin with high encapsulation efficiency, and the nanobowl supported adriamycin liposome was obtained.

The specific synthesis circuit diagram is shown in Figure 8.

1. Preparation of nanobowl supporting liposomes

1 Instruments and materials

1.1 Instrument and equipment

Table 6

XS205s electronic balance	METTLER TOLEDO
JY92-Ⅱ Ultrasonic Cell Crusher	Ningbo Xinzhi Biological Technology Co., Ltd.
HS-70 type thermostatic magnetic stirrer	German IKA company
R-200 Rotary Evaporator	Swiss Buchi Company
Vacuum drying oven	Shanghai Yiheng Technology Instrument Co., Ltd.
Mini extrusion device	Avanti Corporation
Sorvall ST 16 refrigerated centrifuge	Thermo Fisher Scientific
CM-120 Transmission Electron Microscope	Philips Netherlands
Malvern Zetasizer Nano ZS Laser Particle Sizer	Malvern Company
SpectraMax M2 Biomolecular Microplate Reader	American Molecular Instruments Co., Ltd.
ZQTY-70 shaking incubator	Shanghai Zhichu Instrument Co., Ltd.
RLPHR 2-4 LD freeze dryer	U.S. Marin Christ Company
MS2 type vortex mixer	German IKA company
Laboratory ultrapure water system	Millipore Corporation

1.2 Materials and reagents

Table 7

APTES	American Sigma Company
cholesterol	American Sigma Company
HSPC	Avanti Corporation
DSPE-PEG2000	Avanti Corporation
G-50 dextran gel	American GE Company
DiR	Thermo Fisher Scientific
Doxorubicin hydrochloride	Beijing Huafeng Lianbo Technology Co., Ltd.
Trichloromethane	Sinopharm Chemical Reagent Co., Ltd.
Ammonium Sulfate	Shenggong Biological Engineering (Shanghai) Co., Ltd.
Sodium hydroxide (AR grade)	Sinopharm Chemical Reagent Co., Ltd.
Methanol	Sinopharm Chemical Reagent Co., Ltd.

Uranyl acetate	Beijing Zhongjingke Instrument Technology Co., Ltd.
Absolute ethanol	Sinopharm Chemical Reagent Co., Ltd.
hydrochloric acid	Sinopharm Chemical Reagent Co., Ltd.
Glycine	Shenggong Biological Engineering (Shanghai) Co., Ltd.
Triton X-100	Shenggong Biological Engineering (Shanghai) Co., Ltd.
3.5 KD dialysis membrane	Shenggong Biological Engineering (Shanghai) Co., Ltd.

2 Experimental method

2.1 Amination modification of the Nanobowl

1) Take 20 mg of the nanobowl ultrapure water dispersion obtained in the previous preparation, and transfer it to 10 mL of absolute ethanol by ultracentrifugation (30000g, 30min);

2) Add the anhydrous ethanol dispersion containing the nanobowl into a 25mL round bottom flask and stir at a constant speed;

3) Pipette 200μL of APTES into the above-mentioned dispersion and keep stirring at 150rpm overnight;

4) After the reaction is over, take out the reaction solution, remove unreacted APTES by centrifugation (24000g, 30min), collect the precipitate and disperse it in 5mL ultrapure water, this process is repeated 3 times;

5) DLS detects the size and potential of nanoparticles.

2.2 Preparation of Nanobowl Supported Liposomes

1) Precisely weigh 25mg each of cholesterol, HSPC, DSPE-PEG2000, and dissolve them in 1mL of chloroform respectively to prepare a lipid solution of 25mg/mL;

2) Fully dissolve 10 mg of DiR in 1 mL of chloroform;

3) With the mass ratio of HSPC:Chol:DSPE-PEG2000=3:1:1 (if preparing liposomes containing DiR, follow the mass ratio of HSPC:Chol:DSPE-PEG2000:DiR=3:1:1:0.05 (Ratio), pipette an appropriate amount of the above solution so that the total lipid mass is 20 mg, add an appropriate amount of chloroform, dilute to 5 mL, and mix the solution uniformly through an MS2 vortex mixer;

4) Prepare liposomes by thin film dispersion method: add the evenly mixed lipid solution into a 500mL round bottom flask, and connect it to the rotary evaporator, adjust the medium speed, water temperature 40℃, and rotary evaporate for 1h. Remove the chloroform and make the lipid evenly cover the bottom of the round bottom flask;

5) Take off the round bottom flask, put it in a vacuum drying oven, and dry overnight;

6) Prepare 300mM ammonium sulfate solution with pH 5.5: accurately weigh 7.93g of ammonium sulfate and fully dissolve it in 200mL ultrapure water; adjust the pH value to 5.5 with 1M sodium hydroxide solution;

7) Transfer the aminated nanobowl synthesized in 2.1 to the prepared ammonium sulfate solution by centrifugation (24000g, 30min);

8) Add the ammonium sulfate solution containing the aminated nanobowl to the round-bottomed flask after drying overnight, and place it in the ZQTY-70 shaking incubator to shake (250rpm, 60℃) for 1h to fully hydrate the lipids and form a package Vesicles with nanobowls;

9) Fix the liposome vesicle suspension obtained above on the JY92-Ⅱ ultrasonic cell pulverizer, use 35W power, 15 seconds work and 15 seconds pause mode, and ultrasonic probe for 30 minutes to prepare a uniformly dispersed nanobowl Supported liposomes.

2.3 Preparation of ordinary liposomes

1) The thin film dispersion of lipids is prepared by referring to 1)-6) in 2.2;

2) Pipette 5mL of the prepared ammonium sulfate solution, add it to the round bottom flask after drying overnight, and place it in a ZQTY-70 shaking incubator to shake (250rpm, 60℃) for 1h to fully hydrate the lipids and form vesicles Bubble suspension

3) Assemble the liposome mini-extrusion device, install a 200nm pore size PC membrane, and squeeze the obtained vesicle suspension through the extrusion device back and forth 20 times to improve the uniformity of the liposome size.

2.4 Encapsulation of doxorubicin hydrochloride

1) Prepare 10% sucrose dialysate containing 10mM histidine at pH 6.5: Weigh 500g of sucrose, dissolve it in 5L of ultrapure water, add 7.75g of histidine to fully dissolve it; adjust by 1M hydrochloric acid solution pH value to 6.5;

2) Cut two 3.5KD dialysis membranes, add the liposomes supported by the nanobowl prepared in the previous steps and ordinary liposomes respectively, and put the two into a 1L beaker containing sucrose dialysate for dialysis. Change the fluid 4 times during the period;

3) Accurately weigh 2.0 mg of doxorubicin hydrochloride and dissolve it in 1 mL of sucrose dialysate;

4) Add 0.5 mL of 2 mg/mL doxorubicin hydrochloride sucrose solution to the two groups of liposomes after dialysis, and pipette several times to mix thoroughly;

5) Put the mixture of two groups of liposomes and adriamycin hydrochloride into the ZQTY-70 shaking incubator and shake (250rpm, 60℃) for 1h;

6) Put the nanobowl to support the liposomes into the centrifuge, collect the precipitate by centrifugation (24000g, 30min), redisperse it with 10% sucrose dialysate, and repeat the washing three times to remove the free doxorubicin hydrochloride and unencapsulated nano Bowl of liposomes; drop ordinary adriamycin liposomes on the G-50 dextran gel to elute free adriamycin hydrochloride.

2. Establishment of a fluorescence quantitative analysis method for doxorubicin hydrochloride

1. Selection of detection wavelength

1) Weigh out 1 mg of doxorubicin hydrochloride and dissolve it in 10 mL of ultrapure water;

2) Pipette 200μL of sample and drop it into a 96-well plate;

3) Use SpectraMax M2 biomolecule microplate reader to scan the ultraviolet absorption in the range of 200-850nm to obtain the ultraviolet absorption spectrum of adriamycin hydrochloride;

4) Determine the maximum absorption wavelength Dox-max of doxorubicin hydrochloride by ultraviolet absorption spectrum, and use this wavelength as the excitation light wavelength to detect the fluorescence emission spectrum of doxorubicin hydrochloride, and obtain the maximum fluorescence emission wavelength of doxorubicin hydrochloride Em Dox- max.

2. Drawing the standard curve

1) Precisely weigh an appropriate amount of doxorubicin hydrochloride and prepare a stock solution of a certain concentration with ultrapure water, and then continue to dilute the stock solution with ultrapure water to obtain 0.156, 0.313, 0.625, 1.250, 2.500, 5.000, 10.000 μg/mL Doxorubicin hydrochloride reference substance solution;

2) Based on the Dox-max and Em Dox-max obtained by the above method, establish a fluorescence quantitative analysis detection method: use Dox-max as the excitation light wavelength, and detect the above-mentioned adriamycin hydrochloride reference solution at each concentration at the Em Dox-max wavelength的fluorescence emission intensity;

3) Linear regression is performed on the concentration of adriamycin hydrochloride by fluorescence emission intensity to obtain the fluorescence quantitative standard curve equation of adriamycin hydrochloride.

3. Precision

1) Select the above 0.156, 1.250, 10.000 μg/mL doxorubicin hydrochloride reference substance solutions as low-concentration, medium-concentration and high-concentration samples;

2) Detect the fluorescence intensity of each concentration of doxorubicin hydrochloride at 2, 4, and 8 hours to calculate the intra-day standard deviation (RSD Intra-day); perform the detection on 1d, 2d, and 3d to calculate the inter-day standard deviation (RSD Inter -day).

4. Recovery rate

1) Select the above-mentioned 0.156, 1.250, 10.000μg/mL adriamycin hydrochloride reference substance solution each of 3 copies, and carry out fluorescence intensity detection;

2) Bring the obtained fluorescence intensity into the standard curve formula, and calculate the concentration of doxorubicin hydrochloride;

3) The recovery rate is calculated from the measured concentration and the actual concentration.

3. Double fluorescence qualitative detection of nanoparticles

1. Selection of DiR detection wavelength

1) Pipette 20μL of 10mg/mL DiR chloroform solution and dilute to 1mL with methanol;

2) Pipette 200μL of sample and drop it into a 96-well plate;

3) Use SpectraMax M2 biomolecule microplate reader to scan the ultraviolet absorption in the range of 200-850nm to obtain the ultraviolet absorption spectrum of DiR;

4) Determine the maximum absorption wavelength DiR-max of DiR by ultraviolet absorption spectrum, select the wavelength of the excitation light in the ultraviolet absorption region of DiR that has the least influence on the fluorescence spectrum, detect the fluorescence emission spectrum of DiR, and obtain the maximum fluorescence emission wavelength of DiR Em DiR -max.

2. Nanoparticle ultraviolet absorption spectrum and dual fluorescence signal emission spectrum

1) Pipette appropriate amount of Adriamycin-loaded Nanobowl-supported liposomes or ordinary Adriamycin liposomes with DiR embedded in the liposome phospholipid bilayer respectively, and add them to the corresponding 96-well plate;

2) Use SpectraMax M2 biomolecule microplate reader to scan the ultraviolet absorption in the range of 200-850nm to obtain the ultraviolet absorption spectrum of the nanoparticles;

3) Then, according to the aforementioned method, the wavelength of the excitation light in the Dox-max and DiR ultraviolet absorption zone with the least influence on the fluorescence spectrum is selected as the excitation light wavelength, and the doxorubicin hydrochloride and DiR in the nanoparticles are detected. Fluorescence emission spectrum.

4. Determination of liposome encapsulation efficiency and drug loading

1) Precisely pipette 50μL of nanobowl-supported nanobowl-supported liposomes or common adriamycin-supported liposomes loaded with doxorubicin, and dilute to 1mL with ultrapure water;

2) Add 0.2% Triton solution to the sample to lyse the liposomes and release doxorubicin in the internal water cavity;

3) Detect the fluorescence intensity of adriamycin by fluorescence quantitative analysis method;

4) Bring the fluorescence intensity into the standard curve equation to obtain the concentration of doxorubicin and calculate the encapsulation efficiency. The specific formula is as follows:

5) Precisely pipette 1 mL of nanobowl supported doxorubicin liposome or common doxorubicin liposome sample, put it in a constant weight treated EP tube, put it in a -80℃ refrigerator overnight, and then transfer to RLPHR 2-4 Freeze drying in LD freeze dryer.

6) Weigh the freeze-dried sample to calculate the drug loading:

5. Results

1. Characterization of amination modification of nanobowl

After the amination reaction, the DLS results showed that the size of the nanobowl did not change significantly, but its Zeta potential was reversed from the original -30.2±1.1mV to +34.5±1.5mV, as shown in Figure 9.C. The potential flip proves that the surface of the nanobowl has been successfully modified with amino groups, making its Zeta potential positive. At the same time, the results of transmission electron microscopy in Figure 9.A show that the morphology of the nanoparticles has not changed, and the bowl-shaped structure has not been affected.

2. Characterization of nanobowl supported liposomes and ordinary liposomes

With the addition of liposomes, DLS detection showed that the size of the nanobowl supported liposomes increased to 143.6±6.2nm, which is nearly 20nm compared to the size of the nanobowl. At the same time, the result of Zeta potential was -17.9±0.3mV. The ordinary liposomes extruded through a 200nm PC film have a particle size of 148.3±2.9nm and a Zeta potential of -20.3±1.2mV. The nanobowl supports the slight increase in the particle size of liposomes and the reversal of the Zeta potential, which is close to that of ordinary liposomes. Subsequently, the nanoparticle sample negatively stained with uranyl acetate was observed under a transmission electron microscope, and a circle of phospholipid bilayers around the nanobowl could be clearly seen. At the same time, the nanobowl wrapped in the liposome was still clearly visible. The above phenomena have verified the successful encapsulation of liposomes.

3. Ultraviolet absorption spectrum and fluorescence spectrum

3.1 Adriamycin hydrochloride and DiR ultraviolet absorption spectrum and fluorescence spectrum

According to the wavelength scanning results of the microplate reader, the ultraviolet absorption spectra of doxorubicin hydrochloride and DiR were drawn, and the results are shown in Fig. 11, Dox-max=480nm, DiR-max=740nm. Then take 480nm and 700nm as the excitation light wavelengths of the two respectively, and use the microplate reader to obtain the corresponding fluorescence emission spectra, Em Dox-max=580nm, Em DiR-max=780nm.

3.2. Nanoparticle ultraviolet absorption spectrum and dual fluorescence emission spectrum

The UV absorption and fluorescence signals of the prepared two liposomes were measured. The results are shown in Figure 12. Whether it is UV absorption spectrum or fluorescence emission spectrum, the characteristic absorption peaks and emission of adriamycin hydrochloride and DiR can be found. The peak indicates the coexistence of liposomes and doxorubicin in the nanoparticles in the system, which proves the successful entrapment of doxorubicin. Carefully comparing the UV absorption spectra of the NB@DLP group and DLP, it can be found that the absorption of NB@DLP at 300-400nm is significantly higher than that of DLP. It is speculated that this wavelength range is the non-characteristic absorption region of the nanobowl.

4. Determination of nanoparticle encapsulation efficiency and drug loading

4.1. Fluorescence quantitative standard curve of adriamycin hydrochloride

According to the detection result of the microplate reader, record the fluorescence intensity of each concentration of doxorubicin hydrochloride at a wavelength of 580nm, and perform a linear regression of the concentration C (μg/mL) with the fluorescence intensity RFU to obtain RFUDox = 99.11C-9.871, R ² = 0.998. At the same time, the precision and recovery rate of the method were investigated, and the results are shown in Tables 8 and 9. The RSD of intra-day and inter-day precision is less than 3%, indicating that the method has good precision.

Table 8. Precision experiment

Table 9. Recovery rate experiment

4.2. Determination of nanoparticle encapsulation efficiency and drug loading

The fluorescence intensity of doxorubicin hydrochloride in nanoparticles was measured by a microplate reader, and substituted into the standard curve of doxorubicin hydrochloride fluorescence quantitative analysis to obtain the concentration of doxorubicin hydrochloride in the corresponding nanoparticles, and the drug loading and encapsulation efficiency were calculated. The results are shown in Table 10. The results show that the presence or absence of nanobowl support has no significant effect on the drug loading efficiency of liposomes, and the encapsulation efficiency is maintained at around 90%.

Table 10. Nanoparticle encapsulation efficiency and drug loading (n=3)

Six, discussion

1. The effect of nanobowl Zeta potential and particle size on liposome fusion and entrapment

The fusion and entrapment process of nanoparticles and liposomes is the result of many factors. Studies have shown that the Zeta potential and size of the nanoparticles will affect the final fusion result. In the process of fusion and coating of nanoparticles and liposomes, electrostatic adsorption has played a vital role. When the zeta potential of the liposome is negative, if the zeta potential of the nanoparticle is opposite to the positive charge, the liposome and the nanoparticle will quickly approach and adsorb under the action of the electrostatic adsorption force, and finally the nanoparticle and the liposome Fuse and enter the water cavity of the liposome; and when the zeta potential of the nanoparticle is also negative, due to the same electrical property between the nanoparticle and the liposome, the rapid increase in electrostatic repulsion when the two are close will cause the two to fail. Close, thus hindering the fusion of nanoparticles and liposomes. At the same time, the size of the nanoparticles will also affect the fusion with liposomes. When the size of the nanoparticle is much smaller than that of the liposome, although the opposite electrical properties can promote the proximity and adsorption of the nanoparticle and the liposome, the surface potential of the nanoparticle is too small to cause it to interact with the liposome. Fusion, on the contrary, makes a large number of small nanoparticles exist on the surface of liposomes, and these small nanoparticles can continue to adsorb other liposomes, thereby forming cross-links and destroying the dispersion balance of nanoparticles, eventually leading to sedimentation; and when the size of the nanoparticles is When the liposomes are comparable, their surface potential will be sufficient to trigger the fusion of the nanoparticles and liposomes to form a stable combination.

2. Selection of model drug doxorubicin hydrochloride

Doxorubicin hydrochloride, as an anti-tumor treatment drug, belongs to the anthracycline class of broad-spectrum anti-tumor drugs and has strong cytotoxicity. Although doxorubicin hydrochloride is widely used in various anti-tumor treatments, it also brings many adverse reactions due to its strong cytotoxicity. Therefore, pharmacists have been working hard for many years to find effective means to reduce the toxicity of doxorubicin hydrochloride, and preparing it into a corresponding preparation is a dosing regimen. In addition, due to the unique anthracycline structure of doxorubicin, it has strong red fluorescence characteristics, which is convenient for detection and facilitates the design and development of various experiments.

3. The choice of drug delivery method for doxorubicin hydrochloride

When the nanobowl was originally designed to support adriamycin liposomes, it was hoped that a passive drug loading method would be used to directly add a hydration solution with a certain concentration of adriamycin hydrochloride dissolved in the liposome hydration process. However, through experiments, it was found that through this method, the encapsulation rate and loading efficiency of doxorubicin were very low. The main reason is that the volume of the inner water cavity of the liposome is much smaller than the volume of the outer water phase. As a result, most of the doxorubicin hydrochloride stays in the outer water phase and cannot be encapsulated by the liposome. . Therefore, the passive loading method not only fails to achieve high-concentration drug loading, but also causes a large amount of waste of free drugs. For this reason, this article uses a drug loading method similar to the FDA-approved Doxil prescription, and uses the ammonium sulfate gradient method to achieve active loading of doxorubicin hydrochloride. Since doxorubicin hydrochloride is a weakly basic drug, the weak acidity of ammonium sulfate can be used to achieve a higher encapsulation rate. First, hydration is carried out with a hydration solution containing ammonium sulfate, so that the water cavity in the liposome presents a weakly acidic environment; then, the pH gradient between the outer water phase and the inner water cavity is established by dialysis; adriamycin hydrochloride is added to the outer water phase _{, The small NH 3} molecules produced by the ionization balance of ammonium sulfate can easily pass through the phospholipid bilayer and neutralize the doxorubicin hydrochloride in the outer water phase, resulting in the molecularization of doxorubicin and transmembrane into the water cavity of the liposome; After the liposomal water cavity, doxorubicin ^{combines with H +} in the internal water cavity and ionizes again to form a salt; the formed doxorubicin sulfate will form insoluble crystals in the internal water cavity, thereby preventing the re-transmembrane of doxorubicin Leakage to achieve a higher encapsulation rate. The detailed principle is shown in Figure 13.

Seven, summary

By incubating the nanobowl and liposome with ultrasound, the nanobowl supporting liposome is prepared, and the method of active drug loading by ammonium sulfate is used to successfully encapsulate adriamycin. Constructing a nanoparticle characterization evaluation system: DLS detects size and zeta potential changes, transmission electron microscope negative staining observes the phospholipid bilayer structure, microplate reader measures the fluorescence signal of doxorubicin and DiR double-labeled nanoparticles, and performs encapsulation efficiency and Drug loading determination. The final average particle size of the nanoparticles is 143.6nm, and the Zeta potential is -17.9mV. Transmission electron microscopy results indicate that the nanoparticle has complete morphology and clearly visible structure. The successful fusion and coating of the liposome can clearly observe the phospholipid bilayer of the liposome. Nanoparticles detected the ultraviolet absorption and fluorescence signals of doxorubicin and DiR in the microplate reader, which further proved the successful construction of nanoparticles. The encapsulation efficiency and drug loading were 89.57% and 2.34%, respectively. Through active drug loading, the encapsulation rate and drug loading of doxorubicin are significantly improved, which provides a feasibility and theoretical basis for clinical transformation.

Example 3 Investigation of stability of adriamycin liposome supported by nanobowl and evaluation of in vitro anti-tumor treatment

1. Instruments and materials

1. Instrument and equipment

Table 11

XS205s electronic balance	METTLER TOLEDO
JY92-Ⅱ Ultrasonic Cell Crusher	Ningbo Xinzhi Biological Technology Co., Ltd.
HS-70 type thermostatic magnetic stirrer	German IKA company
R-200 Rotary Evaporator	Swiss Buchi Company
Vacuum drying oven	Shanghai Yiheng Technology Instrument Co., Ltd.
Mini extrusion device	Avanti Corporation

Sorvall ST 16 refrigerated centrifuge	Thermo Fisher Scientific
CM-120 Transmission Electron Microscope	Philips Netherlands
Malvern Zetasizer Nano ZS Laser Particle Sizer	Malvern Company
SpectraMax M2 Biomolecular Microplate Reader	American Molecular Instruments Co., Ltd.
ZQTY-70 shaking incubator	Shanghai Zhichu Instrument Co., Ltd.
RLPHR 2-4 LD freeze dryer	U.S. Marin Christ Company
MS2 type vortex mixer	German IKA company
Laboratory ultrapure water system	Millipore Corporation
LSM5 Confocal Laser Microscope	Zeiss, Germany
DP50 upright microscope	Olympus Japan
Carbon dioxide cell incubator	Thermo Fisher Scientific
1300 Series A2 ultra-clean console	Thermo Fisher Scientific

2. Materials and reagents

Table 12

3. Cell lines and animals

4T1 breast cancer cells were purchased from Caliper Life Sciences (Hopkinton, MA). The cell generation number used in this experiment was 3-5 generations.

4. Preparation of related reagents

1) 4T1 cell culture medium: DMEM basic medium, 1 penicillin streptomycin double antibody, 10% fetal bovine serum;

2) 4% paraformaldehyde solution: use PBS (0.01M) with pH 7.4 as the solvent, weigh 40g paraformaldehyde into 800mL PBS solution, shake and dissolve in a constant temperature shaker at 60°C overnight, and then dissolve in the solid paraformaldehyde. After dissolving completely, stop heating, after equilibrating to room temperature, add PBS again to make the volume to 1L.

2. Experimental method

1. Evaluation of stability of doxorubicin liposomes supported by nanobowl

1.1 Evaluation of serum stability

1) Pipette appropriate amount of nanobowl support liposomes and ordinary doxorubicin liposomes loaded with adriamycin, add them to 100% FBS respectively, after pipetting evenly, put them in a constant temperature shaking incubator at 37°C at 120 rpm Uniform oscillation

2) At 0, 2, 4, 6, 8, 12, 24 hours, draw an equal amount of the above mixed solution, and measure the fluorescence intensity Ft of adriamycin in the mixed solution with a microplate reader;

3) After 24 hours, add 0.2% Triton solution to lyse and destroy the liposomes, and after the adriamycin is completely released, the microplate reader records the adriamycin fluorescence intensity F _final ;

4) Calculate the leakage rate of adriamycin in whole serum based on the fluorescence intensity, the detailed formula is as follows:

5) After centrifugation (24000g, 15min), the nanobowl supported adriamycin liposomes at each time point were collected, and the particle size and potential were measured by DLS after redispersion.

1.2. Evaluation of freeze-drying stability

1) Precisely pipette an equal amount of nanobowl supporting doxorubicin liposome or common doxorubicin liposome sample, put it in the ep tube, put it in the -80℃ refrigerator overnight, and then transfer to RLPHR 2-4 LD Freeze drying overnight in a freeze dryer;

2) Take the freeze-dried nanobowl to support adriamycin liposomes or ordinary adriamycin liposomes, add the same amount of ultrapure water again, and vortex to make it fully dispersed;

3) Observe and record the dispersion of nanoparticles;

4) DLS measures the change in the size of nanoparticles after freeze-drying and re-dispersion.

1.3 Evaluation of storage stability

1) Place the prepared nanobowl supported adriamycin liposome dispersion in a refrigerator at 4°C;

2) The particle size and Zeta potential were measured by DLS on the corresponding dates, and the changes in the long-term storage state of the nanoparticles were recorded.

2. 4T1 cell culture

2.1 4T1 cell recovery

1) Take out the frozen 4T1 cells from the liquid nitrogen tank, incubate in a 37℃ water bath, and thaw;

2) After the solid in the cryopreservation tube has melted into liquid, transfer it to the centrifuge tube, centrifuge at 800g for 5 minutes, and discard the supernatant cryopreservation solution;

3) Add 5 mL of cell culture medium containing 10% fetal bovine serum, pipette the cells to make them evenly dispersed, and then put them into a T-25 culture flask;

4) Place the culture flask in _{a constant temperature incubator containing 5% CO 2} at 37° C., and observe the cell growth state under a microscope the next day.

2.2. Cell culture and passage

1) Use a pipette to remove the cell culture fluid in the original T-25 culture flask, and add 1 mL of DPBS to rinse 1-2 times to remove the remaining culture fluid;

2) Add 1mL 0.25% trypsin, shake the culture bottle back and forth to make the trypsin evenly cover the bottom surface of the culture bottle, and incubate it in a constant temperature incubator _{containing 5% CO 2 at 37°C for 3 minutes;}

3) Add 4 mL of cell culture medium containing 10% fetal bovine serum, dilute the trypsin, and terminate the digestion;

4) Use a pipette to pipette the cell suspension to make it evenly dispersed to obtain a single cell suspension. Divide the cell suspension into a new T-25 culture flask at a ratio of 1:4 to supplement fresh cell culture. Liquid to 5mL;

5) Put the culture flask back to 37°C and _{cultivate in a constant temperature incubator containing 5% CO 2} and observe the cell growth state under a microscope the next day;

6) After 2 days, perform the replacement and passage operation again.

2.3 Cell cryopreservation

1) Aspirate the culture fluid in the original T-25 culture flask, add 1 mL of DPBS to rinse 1-2 times to remove the remaining culture fluid;

2) Add 500 μL 0.25% trypsin, shake the culture bottle back and forth to make the trypsin evenly cover the bottom surface of the whole culture bottle, and incubate it in a constant temperature incubator at _{37°C and 5% CO 2 for 3 minutes;}

3) Add 4 mL of cell culture medium containing 10% fetal bovine serum, dilute the trypsin, and terminate the digestion;

4) Pipette the cell suspension to make it evenly dispersed to obtain a single cell suspension;

5) Collect the cell suspension in a sterile centrifuge tube, centrifuge at 800 rpm for 5 minutes, and aspirate the supernatant;

6) Add 1 mL of cell cryopreservation solution and pipette again to form a single cell suspension;

7) Transfer the single cell suspension to a cryopreservation tube, mark it, store it at -80°C overnight, and then transfer it to a liquid nitrogen tank for storage.

2.4 4T1 breast cancer cells uptake experiment of nanobowl supported adriamycin liposomes

1) Prepare nanobowl-supported adriamycin liposomes and ordinary adriamycin liposomes by using the aforementioned method and technology;

2) After digesting 4T1 cells, ^{inoculate them in a small dish with a cover glass at a cell concentration of 2×10 5} cells/mL, with an inoculation volume of 1 mL, and inoculate 3 small dishes in each nanoparticle group;

3) Place the small dish inoculated with cells in a constant temperature incubator at _{37°C and 5% CO 2 for 12 hours;}

4) Add the corresponding liposome dispersion with the adriamycin concentration of 0.5 mg/mL to each small dish, and incubate for 1 hour _{at 37°C, 5% CO 2 constant temperature incubator;}

5) Aspirate the cell culture medium after 1 hour, rinse with DPBS 3 times, and then add 1 mL 4% paraformaldehyde for fixation for 20 minutes;

6) After the fixation, aspirate the paraformaldehyde, rinse again with DPBS 3 times, and finally add 1 mL of DPBS solution containing 6 μL DAPI, incubate for 5 min, and rinse 3 times with DPBS;

7) Place the fixed sample under a laser confocal microscope for observation, select Alexa

The 488 channel observes the uptake of nanoparticles by cells.

3. CCK-8 method to detect cell viability

1) Take 4T1 cells in logarithmic growth phase, dilute with complete cell culture medium to ^{a density of 2×10 5} cells/mL after counting, and inoculate a 96-well plate, add 200 μL to each well. Incubate the culture plate in a constant temperature cell incubator at 37°C and 5% CO _{2 for 12 hours;}

2) Prepare different nanoparticle structures in serum-free culture medium with a drug concentration gradient to prepare a medium containing different concentrations of drug solution, aspirate the old culture solution in the culture plate, and then add the drug solution to each well 200μL freshly prepared culture medium;

3) Place the 96-well plate containing cells again in a 37°C, 5% CO ₂ constant temperature cell incubator for an appropriate time;

4) Dilute the CCK-8 liquid 10 times with serum-free culture medium according to 1:10, aspirate the old culture medium in the culture plate, and add 100 μL of the culture medium containing CCK-8 to each well;

5) Place the culture plate in a 37°C, 5% CO ₂ constant temperature cell incubator and incubate for 0.5-1h;

6) Measure the absorbance of each well at 450nm wavelength with a microplate reader.

4. Statistical processing

The experimental results are presented as "mean±SD", and GraphPad Prism7.0 medical drawing software is used for statistical analysis and graphs are made. The comparison between the two groups was performed by t-test; the comparison between three groups and above was performed by the multivariate analysis of variance. When p<0.05, the difference was considered to be statistically significant.

3. Results

1. Nanoparticle serum stability

In this study, nanoparticles were dispersed in 100% fetal bovine serum and placed in a constant temperature shaking incubator at 37°C to vibrate and cultivate to approximate the surrounding environment of the nanoparticles in the circulatory system and the impact of blood flow. Calculate the leakage rate of doxorubicin by measuring the fluorescence intensity of doxorubicin at different times. The results are shown in Figure 14.A. The leakage rate of doxorubicin liposomes supported by the nanobowl in the serum is significantly reduced. The average leakage rate within 24 hours is 3.34%, which is kept below 5%, and there is almost no leakage. . However, the average leakage rate of ordinary doxorubicin liposomes is 23.00%, and the difference between the two is nearly 7 times. This result suggests that the supporting effect of the nanobowl can effectively reduce the leakage of the drug in the water cavity of the liposome. At the same time, the changes in the particle size and Zeta potential of the liposomes supported by the Nanobowl in the serum within 24 hours were investigated. The results are shown in Figure 14.B, and neither the particle size nor the Zeta potential changed significantly.

2. Nanoparticle freeze-drying stability

After the two liposomes were separately freeze-dried, ultrapure water was added again, and the mixture was vortexed and stirred to make it sufficient. The results showed that after sufficient time of vortexing, the nanobowl supported the adriamycin liposome group can be fully dispersed; however, the ordinary adriamycin liposome group is unevenly dispersed and is in a suspension state. A red precipitate visible to the naked eye was observed at the bottom of the tube, as shown in Figure 15.A,D. Subsequently, the same amount of sample supernatant was drawn for DLS particle size detection, and the Intensity parameter was used as the inspection weight. As a result, it can be found that the particle size distribution range of ordinary adriamycin liposomes has significantly widened, while showing a multimodal distribution; In comparison, the particle size distribution of doxorubicin liposomes supported by the nanobowl still shows a unimodal distribution, with good dispersibility, and the average particle size is similar to that before freeze-drying (Figure 15.B, C, E, F).

3. Storage stability of nanoparticles

The particle size and Zeta potential of the nano-bowl supported adriamycin liposomes stored at 4°C were measured by DLS on a regular basis to monitor the storage stability of the nanoparticles. The results are shown in Figure 16. The nanoparticles maintained stable particle size and Zeta potential for up to 120 days, and the nanoparticles had good dispersion without obvious sedimentation and agglomeration, which proved that the nanoparticles can be stored stably for a long time. .

4. 4T1 cell nanoparticle uptake

Under a laser confocal microscope with a magnification of 40, the uptake of nanoparticles by 4T1 cells was observed with excitation light of the same intensity. It can be seen from Figure 17.A that, as time goes by, the fluorescence intensity of doxorubicin in 4T1 cells gradually increases. It is inferred from this that the uptake of drug-loaded nanoparticles by 4T1 cells also increases with time; at the same time, when After 4 hours of incubation, the fluorescence signal of doxorubicin in the nucleus area was significantly enhanced compared to 1 hour, indicating that doxorubicin began to be released into the nucleus; however, regardless of whether the uptake time was 1 hour or 4 hours, the nanobowl supported the adriamycin liposome and The intracellular fluorescence intensity of ordinary doxorubicin liposomes is basically the same, and there is no significant difference. The fluorescence quantitative statistics are shown in Figure 17.B, which is consistent with the observed phenomenon.

5. The effect of nanomedicine on cell viability

The concentration range of doxorubicin was selected from 0.01-10.00μg/mL for this study, and 0.01, 0.03, 0.1, 0.3, 1, 3, 10μg/mL serum-free medium containing nanoparticle drugs or free drugs were prepared respectively . Add the above-mentioned drug solution to the corresponding 96-well plate and incubate. The incubation time of 48h was selected as the experimental test time point, and the CCK-8 method was performed to detect cell viability. The results are shown in Figure 18.A. First of all, at the in vitro cell level, the free drug group has significantly stronger effects on cell viability than ordinary adriamycin liposomes and nanobowl supported adriamycin liposomes; secondly, After 48 hours of incubation, there was no statistically significant difference between the normal adriamycin liposome group and the nanobowl supported adriamycin liposome group on cell viability. From this, we conclude that at the in vitro cell level, the common adriamycin liposome group and the nanobowl supported adriamycin liposome group have basically the same effect on cell viability, while the free drug is compared with the two groups of liposomes. Has stronger cytotoxicity. In addition, the non-drug-loaded nanobowl-supported liposomes were selected for CCK-8 detection, and the concentration range was 0.01-3 mg/mL and incubated for 48 hours. The results showed no significant cell viability attenuation, indicating the biocompatibility of the nanoparticles Good, there is no toxic effect on tumor cells even at higher doses.

Four, discussion

1. The choice of tumor cells

According to the indications of Doxil approved by the US FDA and Myocet approved for marketing in Europe, adriamycin liposomes are approved for the treatment of metastatic breast cancer, with prolonged survival time and significantly reduced adverse reactions. The growth and metastasis characteristics of mouse 4T1 cells in BALB/c mice are very similar to breast cancer in humans. This tumor cell has been widely used as an animal model of human stage VI breast cancer. Therefore, 4T1 cells were selected as the research object in this example to carry out subsequent experiments.

2. Extracorporeal circulatory system

The circulatory system is the first body environment that is reached after intravenous injection of drugs. In it, nanomedicine will be affected by the interaction of various proteins, cytokines and cells, and will be impacted by the blood flow, resulting in a series of changes. If the circulatory system is simulated in vitro, the ideal state is to collect whole blood from mice for corresponding experiments. However, due to the limitation of the small amount of whole blood in mice and the coagulation of whole blood, this article chooses to use 100% fetal bovine serum The environment of 37°C constant temperature oscillation approximates the interaction of nanomedicine with various proteins and cytokines and the impact of blood flow in the circulatory system of the body, while the interaction with blood cells is not involved in this article.

3. The effect of nano bowls on stability

It can be seen from the foregoing results that the support of the nanobowl successfully reduced the drug leakage of the liposome in the serum environment, and significantly improved the ability of the nanoformulation to redisperse after undergoing freeze-drying. In the circulatory system, the supporting effect of the nanobowl: ① It can offset the impact of part of the blood flow on the liposomes, reduce the deformation degree of the liposomes, thereby reducing the liposome rupture and content leakage caused by excessive deformation ②The combination of various proteins and cytokines in the serum and liposomes, changing the permeability of liposomes is another important reason for the leakage of liposome contents. Due to the special opening structure of the nanobowl, The leakage range is greatly reduced, and only when the permeability of the phospholipid bilayer within the bowl mouth changes, the content will leak, thereby reducing the probability of leakage.

4. The influence of nanobowl lining on cytotoxicity and ability to be ingested

Whether the addition of the nanobowl will make the cells have a certain impact on the uptake capacity of nanomedicine, this question aroused our attention at the beginning of the experimental design. The uptake of nanoparticles by cells is usually related to the surface properties of the nanoparticles, such as the morphology of the nanoparticles, surface groups, and Zeta potential. Although the shape of the nanobowl is irregular, the liposome is completely covered, so that its surface is completely covered by phospholipids and still presents a vesicle shape. Therefore, the nanoparticle morphology, surface groups, and Zeta potential are almost the same as those of ordinary liposomes. The statistical results of the confocal microscope also verified the argument that the lining of the nanobowl does not affect the uptake of liposomes by the cells.

Similarly, whether the inner lining of the nanobowl will affect the drug release behavior of the nanomedicine and affect the cytotoxicity. With this question in mind, the corresponding CCK-8 experiment was designed in this embodiment. The results showed that the nanobowl did not affect the toxicity of drug-loaded liposomes to cells. In addition, the non-medicated blank nanobowl supported liposomes also did not show obvious cytotoxicity, which proved the good biocompatibility of the carrier.

V. Summary

By investigating the changes in the particle size, Zeta potential and leakage rate of the nanomedicine under three different environments and operations in the simulated circulatory system, freeze-drying and 4℃ storage environment, the stability of the nanomedicine was comprehensively evaluated. It can be seen from the above experiments that the prepared nanobowl supported adriamycin liposomes can maintain good dispersibility and extremely low leakage rate regardless of the internal circulation system, freeze-drying and low-temperature storage environments. At the cell level in vitro, the uptake of nano-drugs by 4T1 cells and the cytotoxicity of nano-drugs were investigated. The results showed that the support of the nanobowl did not have a significant effect on the cells, and the uptake of adriamycin liposomes and ordinary adriamycin liposomes for the nanobowl support by the cells and the level of toxicity of the liposomes to the cells were equivalent.

Example 4 In vivo evaluation of nanobowl supporting adriamycin liposome anti-tumor treatment

1. Instruments and materials

1. Instrument and equipment

Table 13

XS205s electronic balance	METTLER TOLEDO
JY92-Ⅱ Ultrasonic Cell Crusher	Ningbo Xinzhi Biological Technology Co., Ltd.
HS-70 type thermostatic magnetic stirrer	German IKA company
R-200 Rotary Evaporator	Swiss Buchi Company
Vacuum drying oven	Shanghai Yiheng Technology Instrument Co., Ltd.
Mini extrusion device	Avanti Corporation
Sorvall ST 16 refrigerated centrifuge	Thermo Fisher Scientific
CM-120 Transmission Electron Microscope	Philips Netherlands
Malvern Zetasizer Nano ZS Laser Particle Sizer	Malvern Company
SpectraMax M2 Biomolecular Microplate Reader	American Molecular Instruments Co., Ltd.
ZQTY-70 shaking incubator	Shanghai Zhichu Instrument Co., Ltd.
MS2 type vortex mixer	German IKA company
Laboratory ultrapure water system	Millipore Corporation
LSM5 Confocal Laser Microscope	Zeiss, Germany
DP50 upright microscope	Olympus Japan
Carbon dioxide cell incubator	Thermo Fisher Scientific
Invitrogen life countess cell counter	Thermo Fisher Scientific
1300 Series A2 ultra-clean console	Thermo Fisher Scientific
Olympus electron microscope	Olympus Japan

2. Materials and reagents

Table 14

DMEM basal medium	Thermo Fisher Scientific
Penicillin double antibody	Thermo Fisher Scientific
Fetal Bovine Serum	Gemini Corporation
DPBS	American Hyclone Company
0.25% pancreatin (with EDTA)	Thermo Fisher Scientific
Paraformaldehyde	Shanghai Aladdin Reagent Co., Ltd.
Normal saline	Huayu (Wuxi) Pharmaceutical Co., Ltd.
Pentobarbital sodium	Merck
DiR probe	Thermo Fisher Scientific
Triton X-100	Shenggong Biological Engineering (Shanghai) Co., Ltd.
Doxorubicin hydrochloride	Beijing Huafeng Lianbo Technology Co., Ltd.
G-50 dextran gel	American GE Company

3. Cell lines and animals

4T1 breast cancer cells were purchased from Caliper Life Sciences (Hopkinton, MA). BALB/c female mice aged 4-6 weeks were provided by Shanghai Slack Laboratory Animal Co., Ltd.

4. Preparation of related reagents

1) 4T1 cell culture medium: DMEM basic medium, 1 penicillin streptomycin double antibody, 10% fetal bovine serum;

2) 4% paraformaldehyde solution: Take PBS (0.01M) with pH 7.4 as the solvent, weigh 40g paraformaldehyde into 800mL PBS solution, shake and dissolve in a constant temperature shaker at 60°C overnight, and then dissolve in solid paraformaldehyde. After dissolving completely, stop heating, after equilibrating to room temperature, add PBS again to make the volume to 1L;

3) 1% sodium pentobarbital anesthetic: weigh 100 mg of sodium pentobarbital, add 10 mL of ultrapure water, vortex until fully dissolved.

2. Experimental method

1. Establishment of 4T1 breast cancer orthotopic vaccination model

1.1 Preparation of 4T1 cell suspension

1) Take a T-75 culture flask containing 4T1 cells, use a pipette to remove the cell culture fluid in the culture flask, and add 5 mL of DPBS to rinse 1-2 times to remove the remaining culture fluid;

2) Add 2mL 0.25% trypsin, shake the culture bottle back and forth to make the trypsin evenly cover the bottom surface of the culture bottle, and incubate it in a constant temperature incubator at _{37°C and 5% CO 2 for 3 minutes;}

3) Add 10 mL of serum-free cell culture medium, dilute the trypsin, and terminate the digestion;

4) Use a pipette to pipette the cell suspension to make it evenly dispersed to obtain a single cell suspension. Take 5μL of the cell suspension and add the same volume of placental blue staining solution. Use the Invitrogen lifecountess cell counter to count the cells of the cell suspension. ；

5) Centrifuge at 800g for 5 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free cell culture medium according to the intracellular concentration calculated before, and gently pipette the cell mass to fully disperse it to obtain single cells Suspension, the final concentration of cell suspension is: 1.6×10 ⁷ cells/mL;

6) Dispense the 4T1 cell suspension into sterile 2mL centrifuge tubes, and refrigerate at 4°C for later use.

1.2 Orthotopic inoculation of 4T1 cells in BALB/c female mice

1) The day before the inoculation, remove the hair on the right abdomen of the mouse and prepare the skin for the next day's operation;

2) On the next day, take skin-treated mice and inject 100μL of 1% pentobarbital anesthetic into the abdominal cavity;

3) After the mouse is completely anesthetized, fix the limbs, wet the right abdomen with normal saline, and use a scalpel to make a light stroke on the abdomen;

4) Use forceps to clamp out the fourth breast fat pad located at the root of the right thigh, and inject 50μl of the 4T1 single cell suspension prepared above;

5) Loosen the forceps, return the fat pad to its position, and suture the abdominal wound;

6) After the inoculation, the mice are returned to the squirrel cage, and the front leg of each mouse is tied with a label tape.

2. Animal grouping and dosing plan

1) 4 days after inoculation, use a vernier caliper to measure the tumor volume: measure the longest and shortest tumor diameters (L, W) every other day, and calculate the tumor volume according to the following formula:

2) After the tumor volume grows to 80mm ³ , according to the tumor volume, evenly divide it into the following 5 experimental groups:

①Saline group (Saline)

②Blank nanobowl supported liposome group (NB@LP)

③Doxorubicin hydrochloride group (Dox)

④Dual adriamycin liposome group (DLP)

⑤Nano bowl support adriamycin liposome group (NB@DLP)

3) On 8th, 11th, and 14th days after vaccination, the mice in each experimental group were injected with the same amount of corresponding drugs into the tail vein. Each drug-containing group was given the drug at a dose of 4 mg/kg of doxorubicin.

3. Monitoring of mouse body weight and tumor volume

1) After grouping the first injection, measure the mouse body weight and tumor length and diameter every 2 days, calculate the tumor volume, and record all the results;

2) Use GraphPad Prism7.0 medical drawing software for data summarization, statistical analysis and chart making.

4. Immunohistochemical staining of tissue sections

1) After the last intravenous administration, select several mice in each experimental group. After execution, the tumor tissues and important organs of the mice were dissected and removed, and they were fully immersed in 4% paraformaldehyde solution for fixation;

2) During the fixation period, replace the fresh 4% paraformaldehyde solution several times to wash away the remaining blood. After the supernatant solution is clarified, embed in paraffin, section, and immunohistochemical staining: TUNEL, PCNA and H&E staining;

3) Observe the stained sections under a microscope, take pictures to collect images, and use Image-Pro Plus 6.0 software for image analysis to calculate the positive rate of tumor cell apoptosis and proliferation.

5. Observation of the survival period of mice

1) After the administration, the mouse body weight and tumor length and diameter are measured every 2 days;

2) Observe the mental condition, vital signs and survival status of mice;

3) When the mouse is dead or the tumor volume exceeds 2000 mm ³ , the death is recorded, and the mouse is euthanized at the same time.

6. Statistical processing

The experimental results are presented as "mean±SD", and GraphPad Prism7.0 medical drawing software is used for statistical analysis and graphs are made. The comparison between the two groups was performed by t-test; the comparison between three groups and above was performed by the multivariate analysis of variance. When p<0.05, the difference was considered to be statistically significant.

3. Results

1. Pharmacodynamic evaluation of nanobowl supported adriamycin liposomes in the treatment of breast cancer

In order to investigate the therapeutic effect of nanobowl-supported adriamycin liposomes on 4T1 breast cancer, BALB/c female mice after orthotopic inoculation with tumor cells were administered one week later, at the end of the day 0, 3, and 6 respectively. Give the corresponding drugs intravenously. From the first administration (day 0), the weight and tumor volume of the mice were recorded and the survival conditions of the mice were observed, and the survival curve was drawn. The results are shown in FIG. 19. It can be seen from Figure 19.B that compared with the control group, the tumor volume growth rate of free adriamycin, ordinary adriamycin liposomes and nanobowl supported adriamycin liposomes all slowed down to varying degrees, indicating Doxorubicin can effectively inhibit tumor growth. However, comparing the three groups of drug-containing experimental groups, it is not difficult to find that according to the order of the anti-tumor effect, the nanobowl supports the adriamycin lipid with the most significant effect, and the tumor almost no longer grows; followed by the ordinary adriamycin lipid. Although the adriamycin liposome group cannot completely inhibit tumor growth like the Nanobowl, the overall growth rate is slow; the worst is the free adriamycin administration group, although the volume growth rate slows down, it is far It is far from reaching the expectation of treating tumors. The reason may be related to the excessively rapid clearance rate of free doxorubicin in the body, which cannot be effectively accumulated in the tumor site. In addition, by monitoring the body weight of mice (Figure 19.C), it was found that during the administration period of the free adriamycin-administered group, the body weight decreased significantly, the body was thinner, and the mental state also appeared to a certain degree of malaise, showing that the free adriamycin group Mycin itself has a certain degree of toxic side effects on the body.

Similarly, Figure 19.D and Table 15 show the survival curve of mice, the physiological saline group, blank nanobowl supported liposomes, free adriamycin, ordinary adriamycin liposomes and nanobowl supported adriamycin liposomes. The median survival time of mice was 24 days, 24 days, 27 days, 30 days and 50 days. Compared with the normal saline control group, the drug-containing administration group prolonged the survival time of tumor-bearing mice to varying degrees; among them, the free adriamycin had little effect compared with the control group, and the intermediate health survival period was only extended by 3 days ( 12.50%); Compared with the control group, the ordinary doxorubicin liposomes were extended by 6 days (25.00%), and the curative effect was improved compared with the free doxorubicin; in the three groups of administration groups, the nanobowl supported the doxorubicin Liposome has the most significant curative effect. The median survival time is the longest, 50 days. Compared with the control group, it is extended by 26 days (108.33%). Compared with ordinary adriamycin liposome, it is extended by 20 days (66.67). %). In addition, within the 72-day observation period, there was still no death in one animal.

Table 15. Median survival period of 4T1 breast cancer mice after administration of different experimental groups

2. Nanomedicine promotes tumor apoptosis and inhibits tumor proliferation

According to the inhibitory effects of the above-mentioned experimental groups on 4T1 tumors, the tumor tissues of tumor-bearing mice were removed, pathological sections and immunohistochemical staining were performed, and TUNEL and PCNA staining were performed to detect tumor cell apoptosis and proliferation. The results are shown in Figure 20.A. The drug-containing experimental group has different degrees of inhibitory effect on tumor cell apoptosis, and also inhibited the proliferation of tumor cells to different degrees; among them, the nanobowl supports the adriamycin lipid The effect of promoting apoptosis and inhibiting growth of the body is the most significant, followed by ordinary adriamycin liposomes, and free adriamycin has the worst effect. The semi-quantitative results are shown in Figure 20.B and C; and the blank nanocarrier There was no significant effect in the group, which was consistent with the results of in vitro cell experiments.

3. Toxicity analysis of nanomedicine to important organs

Similarly, the important organ tissues of tumor-bearing mice were taken, and pathological sections were stained for observation to investigate the effects of different administration groups on the organs of mice. The results are shown in Figure 21. Each experimental group did not cause significant toxicity to the liver, kidney, spleen, and lungs of mice; while free doxorubicin was more toxic to the heart, causing cardiomyocytes to dissolve and break; the nanobowl supported the doxorubicin Liposomes and ordinary doxorubicin liposomes can significantly reduce the toxic and side effects of doxorubicin on the heart, and no obvious lesions are seen in pathological sections; in addition, the blank nanoparticle carrier group is effective for heart, liver, spleen, lung, kidney, etc. No significant toxicity was seen in the tissues, suggesting that the carrier is non-toxic and has good biocompatibility.

Four, discussion

1. The overall therapeutic effect of nanobowl support on breast cancer in vivo

According to the aforementioned experimental results, the support of the Nanobowl can show significantly better efficacy than other experimental groups in the treatment of mouse breast cancer. Whether it is the inhibition of tumor volume or the prolongation of the survival curve of mice, it is more obvious than the other groups. This result is exactly the opposite of the in vitro cell level result of Example 3. At the in vitro cellular level, free adriamycin with the strongest cytotoxicity has the worst effect; while the ordinary adriamycin liposome, which was originally supported by the nanobowl in vitro, is indistinguishable from the adriamycin liposome. The tumor inhibitory effect is not as prominent as the nanobowl supported adriamycin liposome, but it is still slightly better than the free adriamycin; while the nanobowl supported adriamycin liposome has the best tumor suppressing effect , The tumor volume plan no longer grows. The reason is that, first of all, the extremely poor long-circulation characteristics and fast clearance rate of free adriamycin cause a large amount of adriamycin to fail to reach the tumor site, which affects the efficacy of adriamycin; In terms of liposomes, although the entrapment of liposomes can increase its residence time in the circulatory system to a certain extent, experiments have shown that there will still be early drug leakage, which will lead to the drug reaching the tumor site. There is a certain degree of reduction; and the supporting effect of the nanobowl provides a "hard" liner for the liposome, so that it can withstand the impact and destruction of various factors in the circulatory system. Before reaching the tumor site, as much as possible It reduces the leakage of the contents of the drug, so that more doxorubicin will reach the tumor site with the circulatory system, so as to better exert the efficacy of the drug.

2. The impact of nano bowl support on tumors and important organs

Through research on mouse tumor tissues and tissue sections of important organs, it is found that the nanobowl supported adriamycin liposomes can better inhibit tumor cell proliferation and promote tumor cell apoptosis compared with ordinary adriamycin liposomes. , While reducing the side effects of doxorubicin on the heart. The reason may be closely related to the supporting role of the nanobowl. The supporting effect of the nanobowl can reduce the premature leakage of doxorubicin during the circulation process in the body, so that it can carry more drugs to reach the tumor site, thereby improving the efficacy; at the same time, due to the reduction of free drug leakage, the drug in the organs and tissues is reduced. The concentration is reduced, thereby reducing the toxicity to other organs.

V. Summary

In this paper, the orthotopic inoculation 4T1 breast cancer model was successfully constructed on BALB/c female mice, and different drugs were injected into the tail vein of tumor-bearing mice to observe the therapeutic effects of each group of drugs. Through the detection of mouse body weight, tumor volume, and mouse survival curve, the overall macroscopic efficacy of the mouse after administration is evaluated; the tumor tissue and important organs are analyzed by immunohistochemical staining to investigate the apoptosis and growth of tumor tissue And organ necrosis, further analyze and evaluate the effect of anti-tumor treatment from a microscopic point of view. The results showed that the overall anti-tumor effect of nanobowl supported adriamycin liposomes was the best, followed by ordinary adriamycin liposomes, while the free adriamycin group had poorer efficacy, and the weight of mice decreased significantly during the administration period. Trends, H&E results also show that it is more toxic to the heart.

Example 5 Preparation method of nanobowl supported irinotecan/vincristine liposomes

The preparation method is basically the same as that of Example 1-2. You can refer to Example 1-2, except that the doxorubicin in it is replaced with irinotecan or vincristine.

Example 6 Nanobowl supporting irinotecan/vincristine liposome effect experiment

1. Experimental method:

In order to detect membrane instability and leakage of irinotecan or vincristine caused by serum, irinotecan or vincristine liposomes or nanobowl supported liposomes were dispersed in pure at 37°C. In FBS (fetal bovine serum), shake at a speed of 200 rpm. After a certain period of time, the liposomes are purified with a Zebaspin desalting column (Thermo-Scientific) to remove irinotecan or vincristine outside the liposomes. The drug-retaining liposomes were mixed with 9 times the volume of 0.75M HCL (containing 90% isopropanol), and centrifuged. The supernatant containing irinotecan or vincristine is volatilized in a SpeedVac concentrator (Thermo Scientific), and the remainder is dissolved in 200 microliters of mobile phase, which is composed of methanol, acetonitrile, water and trifluoroacetic acid , At 24:24:52:0.1 (v/v, pH=3.0), at a flow rate of 1 mL/min, separated by a diamonsil C18 column (4.6×150mm, 5μm, Dikma, China), and detected at 254nm. Calculate the leakage of liposomes in FBS.

2. Experimental results

The results show that compared with ordinary irinotecan liposomes (Irinotecan-LP) and vincristine liposomes (Vincristine-LP), nanobowl supported irinotecan liposomes (NB@Irinotecan-LP) and Changchun The leakage rate of neobase liposomes (NB@Vincristine-LP) is significantly reduced, showing high serum stability.

The invention successfully synthesizes nano particles with a bowl-shaped structure, perfects and optimizes the nano particle synthesis prescription, and establishes a complete set of related characterization and identification methods for the nano bowl. Successfully constructed a nanobowl-supported drug-loaded liposome drug delivery system, explored and established the loading method of doxorubicin hydrochloride/irinotecan/vincristine, and realized the combination of doxorubicin/irinotecan/vincristine High encapsulation rate. Subsequently, the present invention comprehensively explores the stability of the nanobowl supported drug-loaded liposomes, and evaluates its cell uptake behavior and cytotoxicity at the in vitro cell level. Stability test results show that the nanobowl supported drug-loaded liposomes can reduce the leakage rate of drug-loaded liposomes in the circulatory system; at the same time, it can be better redispersed after freeze-drying treatment, which is convenient for preparing freeze-dried powders; In addition, the nanobowl supported adriamycin liposome dispersion can be stored for up to 120 days in an environment of 4°C, without sedimentation or agglomeration, and good dispersibility. The cell test results show that the addition of the nanobowl does not negatively affect the cell uptake behavior and cytotoxicity of drug-loaded liposomes. Finally, the present invention successfully constructed a 4T1 breast cancer orthotopic vaccination model to investigate the therapeutic effect of nanobowl supported adriamycin liposomes on breast cancer. The results showed that the nanobowl supported adriamycin liposomes can effectively inhibit tumor growth and significantly prolong the survival period of tumor-bearing mice; at the same time, compared with the other four groups of experimental groups, the nanobowl supported adriamycin lipids The body can significantly reduce the proliferation of tumor cells and promote the apoptosis of tumor cells; and the results of mouse body weight and organ staining show that the nanobowl supported adriamycin liposome can effectively reduce the toxic side effects of adriamycin and improve mice Quality of Life. The invention discloses a new method for improving the anti-tumor efficacy by improving liposome circulation stability and reducing drug leakage, thereby providing new ideas and theoretical basis for anti-tumor treatment.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be considered. This is the protection scope of the present invention.

Claims (10)

1. The nanobowl supported drug-loaded liposome is characterized in that its preparation method includes the following steps:

   (1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

   (2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

   (3) Preparation of nanobowl-supported drug-loaded liposomes: using the method of active drug loading of ammonium sulfate to encapsulate the drug, the nanobowl-supported drug-loaded liposomes are obtained.
2. The nanobowl supported drug-loaded liposome according to claim 1, wherein the drug in step (3) is selected from any one of doxorubicin, irinotecan, and vincristine.
3. The preparation method of nanobowl supported drug-loaded liposomes according to claim 1, characterized in that it comprises the following steps:

   (1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

   (2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

   (3) Preparation of nanobowl-supported drug-loaded liposomes: using the method of active drug loading of ammonium sulfate to encapsulate the drug, the nanobowl-supported drug-loaded liposomes are obtained.
4. The nanobowl supports doxorubicin/irinotecan/vincristine liposomes, which are characterized in that the preparation method includes the following steps:

   (1) Preparation of nano-bowl: preparing polystyrene nanoparticles → preparing MPS-coated modified polystyrene nanoparticles → preparing peanut-shaped nanoparticles → preparing silica modified peanut-shaped nanoparticles → obtaining nano-bowls;

   (2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

   (3) Nanobowl supported doxorubicin/irinotecan/vincristine liposome preparation: use ammonium sulfate active drug loading method to encapsulate doxorubicin/irinotecan/vincristine to obtain nanobowl support Adriamycin/irinotecan/vincristine liposomes.
5. The nanobowl supported doxorubicin/irinotecan/vincristine liposomes according to claim 4, wherein the polystyrene nanoparticles in step (1) have a particle size of 45-55nm; The peanut-shaped nanoparticles are prepared with styrene monomer and MPS modified polystyrene nanoparticles according to V _styrene :V _MPSNPs =3:1; the silica-modified peanut-shaped nanoparticles are prepared with 0.3g Manufactured by TEOS.
6. The nanobowl supported doxorubicin/irinotecan/vincristine liposomes according to claim 4, wherein the nanobowl supported doxorubicin/irinotecan/vincristine liposomes The average particle size is 140-150nm, and the Zeta potential is -18～-16mV.
7. The preparation method of nanobowl supported adriamycin/irinotecan/vincristine liposomes according to claim 4, characterized in that it comprises the following steps: (1) preparation of nanobowl: preparation of polystyrene nanoparticles → Prepare MPS coated modified polystyrene nanoparticles → prepare peanut-shaped nanoparticles → prepare silica modified peanut-shaped nanoparticles → obtain nano bowls;

   (2) Preparation of empty liposomes supported by the nanobowl: take APTES and the nanobowl prepared in step (1) for amination modification of the nanobowl to obtain an aminated nanobowl → transfer the aminated nanobowl to the prepared nanobowl by centrifugation Obtain the ammonium sulfate solution of the aminated nanobowl from the ammonium sulfate solution in the above, and oscillate it to form nanobowl-wrapped vesicles → perform probe ultrasound on the nanobowl-wrapped vesicles to obtain nanobowl supporting empty liposomes;

   (3) Nanobowl supported doxorubicin/irinotecan/vincristine liposome preparation: use ammonium sulfate active drug loading method to encapsulate doxorubicin/irinotecan/vincristine to obtain nanobowl support Adriamycin/irinotecan/vincristine liposomes.
8. The preparation method according to claim 7, wherein the preparation method of the nanobowl in step (1) is:

   1) Preparation of polystyrene nanoparticles: using styrene as a monomer, SDS as an emulsifier, and KPS as an initiator, the polystyrene nanoparticles are synthesized by emulsion polymerization;

   2) Preparation of MPS-coated modified polystyrene nanoparticles: on the basis of synthesized polystyrene nanoparticles, styrene, MPS and AIBN are added, and MPS-coated modified polystyrene nanoparticles are synthesized through polymerization;

   3) Preparation of peanut-shaped nanoparticles: Mix the prepared MPS-coated modified polystyrene nanoparticles with styrene and VBS in 2), put them in ultrapure water and stir, and use the swelling effect of polystyrene to make MPS The coated modified polystyrene nanoparticles are deformed and broken, and then AIBN is added to initiate the polymerization reaction again, and finally form peanut-shaped nanoparticles;

   4) Preparation of silica-modified peanut-shaped nanoparticles: the peanut-shaped nanoparticles obtained in 3) were ultracentrifugated and then dispersed again in absolute ethanol, and then 25% concentrated ammonia was added, and the configuration contained 50% TEOS After slowly dripping the ethanol solution of, the peanut-shaped nanoparticles modified by silica are obtained;

   5) Preparation of nanobowl: transfer the silica-modified peanut-like nanoparticles obtained in 4) to a rotary evaporator, after evaporating excess ethanol, add tetrahydrofuran to dissolve, collect and precipitate by ultracentrifugation to obtain the final product-nanobowl .
9. The preparation method according to claim 7, wherein the particle size of the polystyrene nanoparticles in step (1) is 50nm; the peanut-shaped nanoparticles are modified polystyrene with styrene monomer and MPS Styrene nanoparticles are prepared according to V _styrene :V _MPSNPs =3:1; the silica-modified peanut-shaped nanoparticles are prepared with 0.3 g of TEOS.
10. Application of the nanobowl supported drug-loaded liposomes of claim 1 in the preparation of anti-tumor drugs.

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